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Weber, F.

Publications and source records attributed to Weber, F..

At least 19 recordsLinked to original sources
bioRxiv2026

Glutamine and GLS1 are key metabolic checkpoints that Limit T cell Infiltration and Function in HNSCC

Head and neck squamous cell carcinoma (HNSCC) is characterized by poor survival and limited benefit from immune checkpoint blockade (ICB), underscoring the need to define targetable mechanisms of immune escape. In a prospective, patient-centered study, we investigated how tumor metabolic reprogramming impairs T cell function and drives ICB resistance. Targeted metabolomics identified two dominant and distinct tumor states: the Warburg phenotype and glutamine deprivation and revealed increased tumor-specific glutamine to glutamate turnover. Integrating spatial metabolic imaging with immunohistochemistry linked these states to immune-cold and immune-excluded tumor architectures. Metabolic states selectively reduced the expression of T cell chemokine receptors highly abundant and prognostic in HNSCC. Accordingly, T cell migration into tumor spheroids was reduced at lower glutamine concentrations. Interfering with glutamine metabolism by pharmacologic inhibition or genetic deletion of glutaminase-1 markedly increased tumor susceptibility to immune cell-mediated cytotoxicity in vitro and ex vivo and sensitized patient-derived tumor fragments (PDTFs) to ICB. This effect extended beyond glutamine restoration and involved disruption of glutathione-dependent ROS defense, impaired tumor proliferation, and increased MHC expression in tumor cells. Consistently, high expression of genes involved in glutamine and glutathione metabolism associated with reduced ICB response in patients. In contrast to HNSCC, other tumor entities including breast cancer show low glutaminase-1 expression. Accordingly, breast cancer PDTFs were not responsive to glutaminase-1 inhibition. Together, these data identify glutaminase-1 as a metabolic checkpoint driving immune escape in HNSCC, and support guided patient stratification for metabolism-targeted immunotherapy, similar to current precision approaches for mutation targeted drugs.

cancer biology↗
bioRxiv2026

TFEB and MCOLN1 are important for Coxiella burnetii egress via lysosomal exocytosis

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C. burnetii spreads to other cell types. While we have information about C. burnetii uptake and the establishment of the replication-competent phagolysosomal-like C. burnetii-containing vacuole (CCV), it is not well studied how C. burnetii exits its host cell. Here, we show that an infection with C. burnetii also triggers the activation of TFEB, a master regulator of autophagy and lysosomal development. The activation occurs in a time-dependent manner and depends on the size of the CCV. Importantly, TFEB activation during C. burnetii infection depend on MCOLN1, which channels Ca2+ across the lysosomal membrane into the cytosol. Knock-down of MCOLN1 resulted in reduced TFEB activation and smaller CCVs, while MCOLN1 activation boosted bacterial egress. Indeed, peripheral CCVs are positive for LAMP1/2 and release bacteria, without inducing host cell death. Importantly, LAMP1/2 and C. burnetii were stainable in non-permeabilized cells at sites of bacterial release, demonstrating fusion of the lysosome with the plasma membrane. Importantly, while replication of C. burnetii is not inhibited in cells lacking LAMP1/2, egress is impaired. Taken together, our data indicates that with increasing CCV size, TFEB is activated by the release of Ca2+ from lysosomes via the MCOLN1 channel, which in turn enables further CCV development and damage of the CCV membrane. This triggers lysosomal exocytosis and egress of C. burnetii without cell death induction.

microbiology↗
bioRxiv2026

Identification of MED13 and DDX60 as critical host factors for SARS-CoV-2 infections

The virus responsible for COVID-19, SARS-CoV-2, continues to spread through the world. The ongoing emergence of new variants with increased viral transmission and immune evasion continue to pose a challenge. Although large-scale genetic screens have identified numerous host factors required for SARS-CoV-2 infection, however the function of these hits remain incompletely understood. In this study, we performed a haploid forward genetic screen in chemically mutagenized mouse embryonic stem cells overexpressing human ACE2 and identified MED13 and DDX60 as important host factors involved in the modulation of SARS-CoV-2 infection. In this study, we have identified and characterized the function of these key element factors for SARS-CoV2 infection. Knockdown of CKM subunits--with the exception of CDK8--or the helicase DDX60 was sufficient to reduce SARS-CoV-2 infection across Vero E6, A549, and Calu-3 cells. During SARS-CoV-2 infection, MED13 was found to function downstream of the JAK/STAT interferon pathway, but also showed another function independently of the interferon response pathway. Surprisingly, while DDX60 is traditionally involved in the interferon response pathway, its knockdown reduces SARS-CoV-2 infection, suggesting DDX60 can promote SARS-CoV-2 infection Interestingly, while inactivation of MED13 or DDX60 markedly reduced SARS-CoV-2 and SARS-CoV infection, it did not affect MERS-CoV. Collectively, these results identify MED13 and DDX60 as critical host determinants for SARS-CoV-2 related-coronaviruses with pandemic potential. Impact statementUsing an unbiased haploid genetic screening approach, this study identifies for the first time MED13 and DDX60 as host factors that influence coronavirus replication through transcriptional and interferon-associated pathways. While DDX60 has previously been linked to antiviral signaling, our findings suggest it can promote coronavirus infection. Most interestingly, the effects of these factors extend beyond SARS-CoV-2 to other coronaviruses. These findings broaden the current understanding of coronavirus-host interaction by highlighting transcriptional control and interferon-associated pathways as modulators of infection rather than focusing solely on viral entry. The work provides mechanistic insight into how host regulatory networks influence coronavirus replication and suggests potential targets for host-directed therapies with activity against multiple present and future coronavirus threats. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. RepositoriesThe data generated in this study are provided in the Supplementary Information/Source Data file. Sequencing data are available on NCBI Sequence Read Archive under the accession number BioProject PRJNA1271794. The next generation sequencing data generated in this study has been deposited in the NCBI Sequence Read Archive (SRA) under accession number SRX29485992, SRX29485993, SRX29485994, SRX29485995, SRX29485996, SRX29485997, SRX29485998, SRX29485999, SRX29486000, SRX29486001, SRX29486002, SRX29486003, SRX29486004, SRX29486005, SRX29486006, SRX29486007, SRX29486010, SRX29486011, SRX29486012, SRX29486013, SRX29486014, SRX29486015, SRX29486016, SRX29486017, SRX29486018, SRX29486019, SRX29486021, SRX29486022, SRX29486023, SRX29486024

microbiology↗
bioRxiv2025

The RNA helicase eIF4A as a novel target in insect cells to combat arboviral infections

Arboviruses transmitted by mosquitoes cause major global health burdens, yet few vaccines or antivirals exist. Targeting host factors required for viral replication offers a promising approach. The DEAD-box RNA helicase eIF4A, a core component of the translation initiation complex eIF4F, unwinds structured 5' UTRs and is critical for many viral RNAs. We tested whether the natural products rocaglates and pateamines, potent eIF4A inhibitors in mammalian cells, also suppress arboviral replication and translation in insect vectors. Silvestrol strongly inhibited Rift Valley fever virus (RVFV) replication in human A549 cells without cytotoxicity, expanding the list of arboviruses dependent on eIF4A. Sequence analysis showed conservation of the rocaglate-binding motif between the arboviral vector Aedes aegypti and the fruit flies Anastrepha suspensa and Drosophila melanogaster. Dual-luciferase assays in insect cells of all three species confirmed silvestrol selectively inhibited translation from purine-rich reporters below cytotoxic thresholds. Purified eIF4A variants retained helicase activity, allowing direct testing of inhibitor interactions. Thermal shift assays demonstrated robust stabilization of eIF4A-RNA complexes by both compound classes in the wildtype proteins, with unexpected rocaglate sensitivity of the putatively insensitive Ae. aegypti H161L mutant, indicating a unique binding pocket geometry of the mosquito protein. Our results identify eIF4A as a conserved and druggable host factor in insects, highlighting its potential as a novel target for transmission-blocking antivirals and insect-specific inhibitors. Author SummaryNatural compounds from the classes of rocaglates and pateamines have shown their potential to inhibit tumor cell growth and to effectively suppress replication of different viruses, including Ebola, corona, picorna, and arboviruses by inhibiting translation initiation through blocking of one of the initiation actors, the helicase eIF4A. eIF4A is needed to unwind structured 5UTRs of selected mRNAs to allow binding of the 43S preinitiation complex. The compounds bind in a pocket of eIF4A, thereby clamping the RNA onto the eIF4A surface. Although eIF4A is highly conserved in eukaryotes not all eIF4A are sensitive to rocaglates. Mainly one key amino acid in the binding pocket determines sensitivity or insensitivity. In this study we expanded the list of arboviruses as potential targets of rocaglate-based drugs. Moreover, we approached the inhibition of arboviral infections from the vector side by studying the interaction between rocaglates and pateamines with eIF4A from the yellow fever mosquito Aedes aegypti, one of the most important arboviral vectors. Our study shows that also the mosquito eIF4A can be targeted by both compound classes. Moreover, our results reveal a unique architecture of the binding pocket that retains sensitivity even upon mutation of the key amino acid.

molecular biology↗
bioRxiv2025

Human cell line tropism, interferon system interactions and NSs mechanisms by the Ntepes and Gabek Forest phleboviruses

Phleboviruses are a large genus within the family Phenuiviridae, class Bunyaviricetes. These arboviruses are present all over the world, and several members can cause severe disease. New viruses are continuously discovered, but rarely isolated and analysed in cell culture to assess their risk to humans. Here, we describe the in cellulo characteristics for two closely related African phleboviruses, Ntepes virus (NTPV) and Gabek Forest virus (GFV), for which human seroprevalence has been reported but pathogenicity remains unknown. For both viruses, human cell lines from liver, lung, and kidney were permissive, and their capacity to suppress and cope with the antiviral type I interferon (IFN) system was comparable to Rift Valley fever phlebovirus MP-12. Consequently, their non-structural protein NSs, a well- known virulence factor of Phenuiviridae, was able to interfere with both the induction and signaling of IFN. Blockade of IFN induction is a conserved NSs activity, and we found that in the case of NTPV and GFV, the NSs targets MAVS (Mitochondrial antiviral signaling protein), a cellular signaling adapter which plays a key role in the induction of IFN gene expression. NTPV and GFV NSs thereby strongly and specifically bind the N-terminal CARD (Caspase recruitment domain) of human MAVS, thus occupying the very domain that is required for MAVS to relay the signal coming from the virus sensor RIG-I. Thus, the cell line tropism, IFN system interactions and NSs mechanisms suggest that NTPV and GFV fulfill the in cellulo criteria for successful infection of humans. ImportanceChikungunyna virus and Zika virus are just two recent examples of arboviruses that have emerged and caused outbreaks in humans. We investigated whether Ntepes virus (NTPV) and Gabek Forest virus (GFV), two closely related bunyaviruses of the genus Phlebovirus with human seroprevalence in certain African regions, have cell culture characteristics that are compatible with a risk for humans. Both viruses were able to infect human cell lines derived from inner organs, and could circumvent the human antiviral interferon system similar to strain MP-12 of the established Rift Valley fever phlebovirus. Specifically, we identified that the interferon antagonists NSs of NTPV and GFV could strongly and specifically bind to a domain in the cellular adaptor protein MAVS that is crucial for the upregulation of the antiviral interferon system. NTPV and GFV may thus have potential to further adapt to humans.

microbiology↗
bioRxiv2025

A century-old museum sample reveals abandavirus with modern day presence in northernEuropean bats

Ancient genome sequences provide invaluable insights into the origin and evolution of viral pathogens, offering a broader temporal perspective that extends well beyond the limited timespan of clinical data, which typically covers at most a few decades. Whereas ancient viral DNA is relatively frequently recovered, ancient viral RNA genomes are scarce due to the fragile nature of RNA molecules. In this study, we explored the feasibility of detecting ancient viral RNA in ethanol-preserved bat samples from a museum collection. We not only detected viral genome fragments but also recovered the coding-complete genome of a bandavirus (species Bandavirus zwieselense, family Phenuiviridae, order Hareavirales, class Bunyaviricetes) from a Common pipistrelle (Pipistrellus pipistrellus) bat collected in northern Germany in 1919. To investigate the modern distribution of Bandavirus zwieselense, we screened bat organs collected in Germany and the Netherlands via RT-qPCR, identifying modern counterparts in nine Common Pipistrelle (P. pipistrellus) bats (collected between 2010 and 2018), and one Serotine bat (Eptesicus serotinus, collected in 1999). The resulting genomic data enabled us to map phylogenetic relationships within this previously uncharacterized virus species (Bandavirus zwieselense) and estimate the timeframe for the most recent common ancestor. Additionally, we performed functional analysis of the S segment encoded nonstructural (NSs) protein of Bandavirus zwieselense in human HEK-293T cells, demonstrating its ability to block interferon induction, a characteristic also observed in the related human-pathogenic Severe fever with thrombocytopenia syndrome virus (SFTSV, species Bandavirus dabieense). This study demonstrates the feasibility of recovering and characterizing viral genomes from ethanol-preserved ancient RNA material, underscoring the significant potential of museum collections to contribute to the understanding of RNA virus evolution.

microbiology↗
bioRxiv2025

JAK inhibitors remove innate immune barriers to allow viral propagation

Janus kinase (JAK) inhibitors are small-molecule therapeutics that reduce inflammation in autoimmune and inflammatory diseases by modulating the JAK-STAT pathway. While effective in alleviating immune-mediated conditions, JAK inhibitors can impair antiviral defences by suppressing interferon (IFN) responses, potentially increasing susceptibility to viral infections. This study investigates the pro-viral mechanism of JAK inhibitors, focusing on baricitinib, across various cell lines, organoids, and viral strains, including a recombinant Rift Valley fever virus, influenza A virus, SARS-CoV-2, and wild type adenovirus. Our findings demonstrate that baricitinib suppresses transcription of IFN-stimulated genes in non-infected cells (ISGs) which is triggered by type I IFNs produced by infected cells, facilitating viral propagation. The pro-viral effects were influenced by viral load, inhibitor concentration, and structural characteristics of the compound. These results underscore the dual effects of JAK inhibitors: reducing inflammation while potentially exacerbating viral infections. Additionally, the findings highlight opportunities to leverage JAK inhibitors for viral research, vaccine production, and drug screening.

immunology↗
bioRxiv2024

A hypothalamic circuit mechanism underlying the impact of stress on memory and sleep

Stress profoundly affects sleep and memory processes. Stress impairs memory consolidation, and similarly, disruptions in sleep compromise memory functions. Yet, the neural circuits underlying stress-induced sleep and memory disturbances are still not fully understood. Here, we show that activation of CRHPVN neurons, similar to acute restraint stress, decreases sleep and impairs memory in a spatial object recognition task. Conversely, inhibiting CRHPVN neurons during stress reverses stress-induced memory deficits while slightly increasing the amount of sleep. We found that both stress and stimulation of CRHPVN neurons activate neurons in the lateral hypothalamus (LH), and that their projections to the LH are critical for mediating stress-induced memory deficits and sleep disruptions. Our results suggest a pivotal role for CRHPVN neuronal pathways in regulating the adverse effects of stress on memory and sleep, an important step towards improving sleep and ameliorating the cognitive deficits that occur in stress-related disorders.

neuroscience↗
bioRxiv2024

Metastasis founder cells activate immunosuppression early in human melanoma metastatic colonization

The earliest steps of lethal metastasis in patients are incompletely understood. To dissect them, we prospectively searched for the earliest detectable disseminated cancer cells (DCC) in sentinel lymph node biopsies of 492 stage I-III patients. By visually-controlled, micromanipulator-assisted isolation and single cell transcriptome analysis of these extremely rare DCC, we identified MCSP+ melanoma cells as strong candidates for metastasis founder cells (MFC) in lymph nodes. Based on a median follow-up time of 6 years, their detection was the strongest predictor of systemic metastasis and death upon multivariable analysis. During transition from single cells to metastasis-initiating clusters, melanoma DCC were exposed to CD8 T cell attack, activated the extracellular vesicular exosomal pathway, and expressed the immunomodulatory proteins CD155 and CD276, but rarely PD-L1. CD155 and CD276-positive extracellular vesicles from patient-derived DCC models exhibited an immunosuppressive activity on CD8 T cells. Our data indicate that either direct targeting of MFC employing MCSP or their immune escape mechanisms might be key for cure of early-stage melanoma.

cancer biology↗
bioRxiv2024

VISION -- an open-source software for automated multi-dimensional image analysis of cellular biophysics

Environment-sensitive probes are frequently used in spectral/multi-channel microscopy to study alterations in cell homeostasis. However, the few open-source packages available for processing of spectral images are limited in scope. Here, we present VISION, a stand-alone software based on Phyton for spectral analysis with improved applicability. In addition to classical intensity-based analysis, our software can batch-process multidimensional images with an advanced single-cell segmentation capability and apply user-defined mathematical operations on spectra to calculate biophysical and metabolic parameters of single cells. VISION allows for 3D and temporal mapping of properties such as membrane fluidity and mitochondrial potential. We demonstrate the broad applicability of VISION by applying it to study the effect of various drugs on cellular biophysical properties; the correlation between membrane fluidity and mitochondrial potential; protein distribution in cell-cell contacts; and properties of nanodomains in cell-derived vesicles. Together with the code, we provide a graphical user interface for facile adoption. We anticipate that VISION will find a broad range of applications in different fields of biology, spanning from molecular and tissue biology to immunology and biophysics. Summary statementVISION, an open-source software, enables high throughput and correlative analysis of cellular biophysical properties.

biophysics↗
bioRxiv2024

Infralimbic activity during REM sleep facilitates fear extinction memory

Rapid eye movement (REM) sleep is known to facilitate fear extinction and play a protective role against fearful memories. Consequently, disruption of REM sleep after a traumatic event may increase the risk for developing PTSD. However, the underlying mechanisms by which REM sleep promotes extinction of aversive memories remain largely unknown. The infralimbic cortex (IL) is a key brain structure for the consolidation of extinction memory. Using calcium imaging, we found in mice that most IL pyramidal neurons are intensively activated during REM sleep. Optogenetically suppressing the IL activity during REM sleep within a 4-hour window after auditory-cued fear conditioning impaired extinction memory consolidation. In contrast, REM-specific inhibition of the IL cortex after extinction learning did not affect the extinction memory. Whole-cell patch-clamp recordings demonstrated that inactivating IL neurons during REM sleep depresses their excitability. Together, our findings demonstrate that REM sleep after fear conditioning facilitates fear extinction by enhancing IL excitability, and highlight the importance of REM sleep in the aftermath of traumatic events for protecting against traumatic memories.

neuroscience↗
bioRxiv2023

Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

neuroscience↗
bioRxiv2023

Preserved particulate organic carbon is likely derived from the subsurface sulfidic photic zone of the Proterozoic Ocean: evidence from a modern, oxygen-deficient lake

I-AbstractBiological processes in the Proterozoic Ocean are often inferred from modern oxygen-deficient environments (MODEs) or from stable isotopes in preserved sediment. To-date, few MODE studies have simultaneously quantified carbon fixation genes and attendant stable isotopic signatures. Consequently, how carbon isotope patterns reflect these pathways has not been thoroughly vetted. Addressing this, we profiled planktonic productivity and quantified carbon fixation pathway genes and associated carbon isotope values of size-fractionated (0.2 - 2.7 and > 2.7 m) particulate organic carbon values (813CPOC) from meromictic Fayetteville Green Lake, NY, USA. The high-O2 Calvin-Benson-Bassham (CBB) gene (cbbL) was most abundant in the <2.7 m size fraction in shallow oxic and deep hypoxic waters, corresponding with cyanobacterial populations. The low-O2 CBB gene (cbbM) was most abundant near the lower oxycline boundary in the larger size fraction, coincident with purple sulfur bacteria populations. The reverse citric acid cycle gene (aclB) was equally abundant in both size fractions in the deepest photic zone, coinciding with green sulfur bacteria populations. Methane coenzyme reductase A (mcrA), of anaerobic methane cyclers, was most abundant at the lower oxycline boundary in both size fractions, coinciding with Methanoregula populations. 813CPOC values overlapped with the high-O2 CBB fixation range except for two negative excursions near the lower oxycline boundary, likely reflecting assimilation of isotopically-depleted groundwater-derived inorganic carbon by autotrophs and acetate oxidation by sulfate-reducers. Throughout aphotic waters, 813CPOC values of the large size fraction became 13C-enriched, likely reflecting abundant purple sulfur bacterial aggregates. Microalgal-like isotopic signatures corresponded with increases in cbbL, cbbM and aclB, and enrichment of exopolymer-rich prokaryotic photoautotrophs aggregates. Results suggest that 813CPOC values of preserved sediments from areas of the Proterozoic Ocean with sulfidic photic zones may reflect a mixture of alternate carbon-fixing populations exported from the deep photic zone, challenging the paradigm that sedimentary stable carbon isotope values predominantly reflect oxygenic photosynthesis from surface waters.

microbiology↗
bioRxiv2023

Identification of host factor for Rift valley fever Phlebovirus

BackgroundRift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. MethodologyTo identify the host factors or genes essential for RVFV replication, we conducted a CRISPR-Cas9 knock-out screen in human A549 cells. We then validated the putative genes using siRNA-mediated knockdowns and CRISPR-Cas9-mediated knockout studies, respectively. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers by plaque assay or TCID50 assay. FindingsWe identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knockdowns found that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of LRP1 in RVFV replication was previously described in detail. Knock-out A549 cell lines were generated and used to dissect the effect of WRD7 on RVFV and another bunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knock-out cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24h) when compared to LACV which was affected an earlier replication phase (12h). ConclusionIn summary, we have identified WDR7 as an essential host factor for the replication of two relevant bunyaviruses, RVFV and LACV. Future studies will investigate the mechanistic role by which WDR7 facilitates Phlebovirus replication. Authors SummaryRift Valley fever phlebovirus is a high consequence pathogen that infects multiple animal species and also humans. Currently, there are no control measures available to treat RVF in humans and to prevent the incursion of Rift Valley fever virus into non-endemic countries. RVFV poses a significant threat to animal and human health in countries where it is endemic. RVFV replication depends on the hosts machinery to complete its replication cycle. Therefore, one way to control virus replication is to disrupt the interaction between the virus and the host proteins important for replication. In this study, we identified a host factor, the WDR7 gene, that is critical for RVFV replication. The identification of this host factor is important as it can potentially lead to the development of antiviral strategies to control Rift Valley fever in both humans and animals.

microbiology↗
bioRxiv2023

Homeostatic regulation of REM sleep by the preoptic area of the hypothalamus

Rapid-eye-movement sleep (REMs) is characterized by activated electroencephalogram (EEG) and muscle atonia, accompanied by vivid dreams. REMs is homeostatically regulated, ensuring that any loss of REMs is compensated by a subsequent increase in its amount. However, the neural mechanisms underlying the homeostatic control of REMs are largely unknown. Here, we show that GABAergic neurons in the preoptic area of the hypothalamus projecting to the tuberomammillary nucleus (POAGAD2[->]TMN neurons) are crucial for the homeostatic regulation of REMs. POAGAD2[->]TMN neurons are most active during REMs, and inhibiting them specifically decreases REMs. REMs restriction leads to an increased number and amplitude of calcium transients in POAGAD2[->]TMN neurons, reflecting the accumulation of REMs pressure. Inhibiting POAGAD2[->]TMN neurons during REMs restriction blocked the subsequent rebound of REMs. Our findings reveal a hypothalamic circuit whose activity mirrors the buildup of homeostatic REMs pressure during restriction and that is required for the ensuing rebound in REMs.

neuroscience↗
bioRxiv2023

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurB and MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs and provide useful enzymatic tools for the potential use in and development of UDP-GlcNAc capture and sequencing protocols.

biochemistry↗
bioRxiv2023

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e. for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a valuable model to study the maintenance of a high transcriptional upregulation. SUMMARY STATEMENTThe thyroglobulin gene is highly and permanently expressed in thyrocytes of all vertebrates, at any condition and round the clock, offering a unique model to study mechanisms of high upregulation maintenance and chromatin dynamics

cell biology↗