bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogenicity and protective efficacy of an influenza virus-like particle-based SARS-CoV-2 hybrid vaccine candidate in rhesus macaques
Authors: Rahman Sheikh Abdul , Bommireddy Ramireddy , Routhu Nanda Kishore , Lai Lilin , Pack Christopher D. , Ramachandiran Sampath , Suthar Mehul S. , Reddy Shaker J. C. , Selvaraj Periasamy , Amara Rama Rao
Published: May 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus co-infections present a heightened COVID-19 disease and hospitalization cases. Here, we studied the immunogenicity and efficacy of an influenza-A/PR8 virus-like particle (FluVLP)-based hybrid vaccine candidate displaying GPI-anchored SARS-CoV-2 receptor binding domain fused to GM-CSF and GPI-anchored interleukin-12 (FluVLP-RBD) in rhesus macaques. Animals (n=4/group) received two doses of eitherFluVLP orFluVLP-RBD vaccine four weeks apart and were challenged with SARS-CoV-2 (WA1/2020) infection via intranasal and intratracheal routes. We determined vaccine-induced IgG and neutralizing antibody titers in serum and their association with viral replication in the lower and upper airways (lung, throat, and nose) and lung-associated pathologies.FluVLP-RBD vaccine induced a strong binding IgG in serum against multiple SARS-CoV-2 variants (WA1/2020, Delta and Omicron; BA.1). Both vaccines induced strong influenza A/PR8-specific IgG. Following the SARS-CoV-2 challenge, all four animals in theFluVLP-RBD group showed a profound control of virus replication in all three airway compartments as early as day 2 through day 10 (day of euthanasia). This level of viral control was not observed in theFluVLP group as 2-3 animals exhibited high virus replication in all three airway compartments. The protection in theFluVLP-RBD vaccinated group correlated positively with post challenge neutralizing antibody titer. These results demonstrated that aFluVLP-based hybrid SARS-CoV-2 vaccine induces strong antibody responses against influenza-A/PR8 and multiple SARS-CoV-2 RBD variants and protects from SARS-CoV-2 replication in multiple compartments in macaques. These findings provide important insights for developing multivalent vaccine strategies for respiratory viruses.

Importance

Co-infection with multiple respiratory viruses poses a greater risk than individual infections, especially for individuals with underlying health conditions. Studies in humans consistently demonstrated that simultaneous infection with SARS-CoV-2 and influenza leads to more severe respiratory illness and an increased rate of hospitalization. Therefore, developing hybrid vaccines targeting multiple respiratory viruses is of high importance. The hybrid vaccines also help to reduce the economic and logistic burden associated with vaccine coverage, distribution and storage. Here, we evaluate the immunogenicity and effectiveness of a novel hybrid flu-SARS-CoV-2 vaccine candidate using a nonhuman primate pre-clinical model. Our findings reveal that this vaccine elicits a strong immune response against influenza and SARS-CoV-2 viruses. Importantly, it provides strong protection against SARS-CoV-2 infection and associated pathological conditions.
Rapidly inducible yeast surface display for antibody evolution with OrthoRep
Authors: Paulk Alexandra M. , Williams Rory L. , Liu Chang C.
Published: May 2024

We recently developed ‘autonomous hypermutation yeast surface display’ (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence activated cell sorting (FACS). AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 hours of induction. Here, we report an updated version of the AHEAD platform that utilizes a synthetic β-estradiol induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system as well as traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.
The mammary glands of cows abundantly display receptors for circulating avian H5 viruses
Authors: Ríos Carrasco María , Gröne Andrea , van den Brand Judith M.A. , de Vries Robert P.
Published: May 2024

Influenza A viruses (IAV) from the H5N1 2.3.4.4b clade are circulating in dairy farms in the United States of America, and goat infections have also been reported. These ruminants were presumed not to be hosts for IAVs. Previously, IAV-positive mammalian species were hunters and scavengers, possibly getting infected while feeding on infected birds. It is now presumed that H5N1 viruses that circulate in US dairy cattle transmit through a mammary gland route, in contrast to transmission by aerosols via the respiratory tract, although the latter cannot be excluded. The receptor display in the mammary and respiratory tract in cows for IAVs is unknown. Here, we used recombinant HA proteins representing current circulating and classical H5 viruses to determine the distribution of IAV receptors in the respiratory and mammary tract tissues of cows and goats. Most of the sialome of the cow and goat respiratory tract is lined with sialic acid modifications such as N-glycolyl and O-acetyl, which are not bound by IAV. Interestingly, the H5 protein representing the cow isolates bound significantly in the mammary gland, whereas classical H5 proteins failed to do so. Furthermore, whereas the 9-O-acetyl modification is prominent in all tissues tested, the 5-N-glycolyl modification is not, resulting in the display of receptors for avian IAV hemagglutinins. This could explain the high levels of virus found in these tissues and milk, adding supporting data to this possible virus transmission route.
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

Aspergillus 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.

IMPORTANCE

The 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

Human 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 SUMMARY

Human 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

The 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

Gene 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

Coronaviruses 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

Protein 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.
Early-life glucocorticoids accelerate lymphocyte count senescence in roe deer
Authors: Lalande Lucas D. , Bourgoin Gilles , Carbillet Jeffrey , Cheynel Louise , Debias François , Ferté Hubert , Gaillard Jean-Michel , Garcia Rebecca , Lemaitre Jean-François , Palme Rupert , Pellerin Maryline , Peroz Carole , Rey Benjamin D. , Vuarin Pauline , Gilot-Fromont Emmanuelle
Published: May 2024

Immunosenescence corresponds to the progressive decline of immune functions with increasing age. Although it is critical to understand what modulates such a decline, the ecological and physiological drivers of immunosenescence remain poorly understood in the wild. Among them, the level of glucocorticoids (GCs) during early life are good candidates to modulate immunosenescence patterns because these hormones can have long-term consequences on individual physiology. Indeed, GCs act as regulators of energy allocation to ensure allostasis, are part of the stress response triggered by unpredictable events and have immunosuppressive effects when chronically elevated. We used longitudinal data collected over two decades in two populations of roe deer (Capreolus capreolus) to test whether higher baseline GC levels measured within the first year of life were associated with a more pronounced immunosenescence and parasite susceptibility. We first assessed immunosenescence trajectories in these populations facing contrasting environmental conditions. Then, we found that juvenile GC levels can modulate lymphocyte trajectory. Lymphocyte depletion was accelerated late in life when FGMs were elevated early in life. Although the exact mechanism remains to be elucidated it could involve a role of GCs on thymic characteristics. In addition, elevated GC levels in juveniles were associated with a higher abundance of lung parasites during adulthood for individuals born during bad years, suggesting short-term negative effects of GCs on juvenile immunity, having in turn long-lasting consequences on adult parasite load, depending on juvenile environmental conditions. These findings offer promising research directions in assessing the carry-over consequences of GCs on life-history traits in the wild.
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

Beta-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.

IMPORTANCE

Amoxicillin 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

The 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

Influenza 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

B 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

Pioneer 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

Background

Nitric 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.

Methods

eNOS 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.

Results

eNOS 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.

Conclusions

Our 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.
Quantifying photosynthetic restrictions
Authors: Bellasio Chandra
Published: May 2024

Quantifying the effect of factors controlling CO2assimilation is crucial for understanding plant functions and developing strategies to improve productivity. Methods exist in numerous variants and produce various indicators, such as limitations, contributions, and sensitivity, often causing confusion. Simplifications and common mistakes lead to overrating the importance of diffusion – whether across stomata or the mesophyll. This work develops a consistent set of definitions that integrates all previous methods, offering a generalised framework for quantifying restrictions. Ten worked examples are provided in a free downloadable spreadsheet, demonstrating the simplicity and applicability to a wide range of questions.
Differential TLR-ERK1/2 activity promotes viral ssRNA and dsRNA mimic-induced dysregulated immunity in macrophages
Authors: Shrestha Rakshya , Johnson Paige , Ghimire Roshan , Whitley Cody , Channappanavar Rudragouda
Published: May 2024

RNA virus induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as ‘dysregulated immunity,’ is caused by viral single-stranded RNA (ssRNA) and double-stranded-RNA (dsRNA) mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We show that murine bone marrow-derived macrophages (BMDMs) stimulated with TLR3 and TLR7 agonists induce differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified a significantly higher cell-surface and endosomal TLR7 expression compared to TLR3, which further correlated with early and robust MAPK (pERK1/2 and p-P38) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2, p38, and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA-mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG responses, and blocking ERK1/2 activity would mitigate viral-RNA/TLR-induced dysregulated immunity.
Insights into Human Norovirus Cultivation in Human Intestinal Enteroids
Authors: Ettayebi Khalil , Kaur Gurpreet , Patil Ketki , Dave Janam , Ayyar B. Vijayalakshmi , Tenge Victoria R , Neill Frederick H. , Zeng Xi-Lei , Speer Allison L. , Di Rienzi Sara C. , Britton Robert A. , Blutt Sarah E. , Crawford Sue E. , Ramani Sasirekha , Atmar Robert L. , Estes Mary K.
Published: May 2024

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells into intestinal organoids that were transplanted and matured in mice before making enteroids (H9tHIEs), genetically-engineered (J4FUT2knock-in [KI], J2STAT1knock-out [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KIHIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.

Importance

HuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from i) different intestinal segments of single donors, ii) human embryonic stem cell-derived organoids transplanted into mice, iii) genetically-modified lines, and iv) a patient with chronic variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically-modified J4FUT2-KIHIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.
A proposal for the quantum mechanical study of genomic mutations driven by environmental stressors
Authors: Mejía-Díaz Héctor , Santiago-Alarcon Diego , Venegas-Andraca Salvador E.
Published: May 2024

We propose a novel quantum statistical method to characterize DNA point mutations under the influence of UVC radiation, salinity, and temperature. We consider an open quantum system composed of an external environment — high-energy photons — coupled to the DNA molecule, and using energy considerations we estimate free parameters in a many-body Hamiltonian, to characterize critical behaviour in the system. The model presented here offers the advantage that one does not explicitly need to know each detail of the interaction between the base-pairs and the environment, by knowing whether the effect is to associate or dissociate the DNA molecule, one can incorporate it into the model.

We show that by fine-tuning the free parameters, the model gives results that are within a biologically realistic range in the energetic scale. Importantly, specific heat values show that by coupling DNA to an external bath, the system dynamics leads to larger fluctuations at slightly lower temperatures. Following this research strategy, one could use experimental data to extract a correlation between specific heat and DNA changes, which would provide insight to predicting evolutionary changes.
TGF-β Signaling in Cranial Neural Crest Affects Late-Stage Mandibular Bone Resorption and Length
Authors: Houchen Claire J. , Ghanem Saif , Kaartinen Vesa , Bumann Erin Ealba
Published: May 2024

Malocclusions are common craniofacial malformations which cause quality of life and health problems if left untreated. Unfortunately, the current treatment for severe skeletal malocclusion is invasive surgery. Developing improved therapeutic options requires a deeper understanding of the cellular mechanisms responsible for determining jaw bone length. We have recently shown that neural crest mesenchyme (NCM) can alter jaw length by controlling recruitment and function of mesoderm-derived osteoclasts. Transforming growth factor beta (TGF-β) signaling is critical to craniofacial development by directing bone resorption and formation, and heterozygous mutations in TGF-β type I receptor (TGFBR1)are associated with micrognathia in humans. To identify what role TGF-β signaling in NCM plays in controlling osteoclasts during mandibular development, mandibles of mouse embryos deficient in the gene encodingTgfbr1specifically in NCM were analyzed. Our lab and others have demonstrated thatTgfbr1fl/fl;Wnt1-Cremice display significantly shorter mandibles with no condylar, coronoid, or angular processes. We hypothesize that TGF-β signaling in NCM can also direct later bone remodeling and further regulate late embryonic jaw bone length. Interestingly, analysis of mandibular bone through micro-computed tomography and Masson’s trichrome revealed no significant difference in bone quality between theTgfbr1fl/fl;Wnt1-Cremice and controls, as measured by bone perimeter/bone area, trabecular rod-like diameter, number and separation, and gene expression of Collagen type 1 alpha 1 (Col1α1) and Matrix metalloproteinase 13 (Mmp13). Though there was not a difference in localization of bone resorption within the mandible indicated by TRAP staining,Tgfbr1fl/fl;Wnt1-Cremice had approximately three-fold less osteoclast number and perimeter than controls. Gene expression of receptor activator of nuclear factor kappa-β (Rank) andMmp9, markers of osteoclasts and their activity, also showed a three-fold decrease inTgfbr1fl/fl;Wnt1-Cremandibles. Evaluation of osteoblast-to-osteoclast signaling revealed no significant difference betweenTgfbr1fl/fl;Wnt1-Cremandibles and controls, leaving the specific mechanism unresolved. Finally, pharmacological inhibition ofTgfbr1signaling during the initiation of bone mineralization and resorption significantly shortened jaw length in embryos. We conclude that TGF-β signaling in NCM decreases mesoderm-derived osteoclast number, that TGF-β signaling in NCM impacts jaw length late in development, and that this osteoblast-to-osteoclast communication may be occurring through an undescribed mechanism.
Establishing remote communication between human operator and engineered intestinal microbe via bio-electronic optical language
Authors: Zhang Xinyu , Feng Zhijie , Li Lianyue , Yang Haoyan , Dai Pengxiu , Yang Chao , Sun Dawei , Li Hongxiang , Wang Hanxin , Xue Huimin , Wang Yaxin , Liu Xinyu , Li Mingshan , Lu Shenjunjie , Ma Xiaoyan , Liu Jing , Shi Yanyan , Du Taofeng , Liu Duo , Wang Hanjie
Published: May 2024

Synthetic biology enables microbes to be engineered to detect biomarkers and deliver therapies in situ, offering advantages such as self-replication and localized response and treatment. However, the engineered microbes in the intestine remains a "black box", the status of microbial functionality is inaccessible, and the activation of microbial functions is uncontrollable. There is a demand of a "language" between microbes and human for real-time and in-situ monitoring and controlling engineered microbes in the intestine. Light, due to its advantages of specificity and accuracy in the body, was chosen as the language to establish an interactive communication path between human and the microbes with the features of sophisticated monitoring and controlling. For effectively remote operation of this "language" in the intestine, we introduced an electronic capsule as the intermedia, translating information and instructions understandable for human operator into bacterial regulatory and detectable signals in vivo. In this paper, the electronic capsule and the engineered Escherichia coli Nissle 1917 (EcN) strains were designed in a collaborative manner. An EcN-to-capsule bioluminescent signal transmission was established and testified as the language to report the status of engineered EcN, especially the response of EcN to intestinal environment. Similarly, another capsule-to-EcN optogenetic signal transmission was established and testified as the language to control the function of engineered EcN, especially the secretion of therapeutic substances. On this basis, bidirectional communication between human operator and intestinal microbes was achieved via bidirectional bio-electronic optical language. The communication was established to monitor and regulate engineered EcN community in the intestine in 50-90 kg live pigs. The in vitro and in vivo estimations demonstrated the capabilities of monitoring and regulating the engineered microbes, offering a remotely controllable language-based communication between human operator and internal intestinal microbes.
Dorsal striatum involvement in response conflict management – a lesion study in rats
Authors: Poitreau Julien , Burle Boris , Sargolini Francesca
Published: May 2024

Action control allows to respond to relevant stimuli while ignoring the non-relevant stimuli in the surrounding environment. In humans this process is generally studied in conflict tasks, such as the Simon task, in which participants respond with a left or right button press to the non-spatial relevant feature (e.g. the color) of a lateralized stimulus, while ignoring the stimulus position. In this study we used a visual version of the Simon task that we have previsously developed in rats to investigate the involvement of the dorsal striatum, a brain area that is central in action control processes. We tested the effect of excitotoxic lesions of the dorsomedial (DMS) and dorsolateral (DLS) areas in learning to control response interference. We showed that both DMS and DLS lesions negatively impacted rat performances, and this effect strongly depends on task practice. These results suggest an involvement of both areas in learning to manage response conflict.
Review of how the generalized regression estimators contribute to estimating the financial and economic data with missing observations under unequal probability sampling
Authors: Lawson Nuanpan
Published: May 2024

Purpose

Knowing financial and economic information beforehand benefits in planning and developing policies for every country especially for a developing country like Thailand and for other Asian countries. Unfortunately, missing data or non-response plays an essential role in many areas of studies including finance and economics. Eradication of missing data in a proper way before further analysis can gain remarkable outcomes and can be effective for planning policies. This review on the generalized regression estimators for population total can be applied to financial, economic and other data when missing data are present.

Design/methodology/approach

The generalized regression estimators for estimating population total, including the variance estimators under unequal probability sampling without replacement with missing data are explored under the reverse framework. Applications to financial and economic data in Thailand are also reviewed.

Findings

The review of literatures related to the proposed estimator shows the best performance, giving smaller variances in all scenarios.

Originality/value

The generalized regression estimators can assist in estimating financial and economic data that contain missing values with different missing mechanisms and can be used in other applications which help gain more superior estimators.