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Abstract Previous studies of natural variants in Drosophila melanogaster implicated the Wnt signaling receptor frizzled in sleep. Given that the Wnt signaling pathway is highly conserved across species, we hypothesized that frizzled class receptor 1 (Fzd1), the murine homolog of frizzled, would also have a role in sleep. Using a CRISPR transgenic approach, we removed most of the Fzd1 coding region from C57BL/6N mice. We used a video assay to measure sleep characteristics in Fzd1-deficient mice. As Wnt signaling is known to affect visuospatial memory, we also examined the impact of the deletion on learning and memory using the novel object recognition (NOR) paradigm. Fzd1-deficient mice had altered sleep compared to littermate controls. The mice did not respond differently to the NOR paradigm compared to controls but did display anxiety-like behavior. Our strategy demonstrates that the study of natural variation in Drosophila sleep translates into candidate genes for sleep in vertebrate species such as the mouse.

Down syndrome (DS) is a common genetic condition affecting people worldwide. It involves cognitive disabilities for which there are no drug therapies. The Ts65Dn mouse model of DS shows cognitive impairment due to a reduction in neuron number and connectivity as well as excessive neuronal activity, as γ-aminobutyric acid (GABA) antagonist treatment restores memory in these mice. Our study showed the effects of GABA antagonist treatment on sleep and decision-making in Ts65Dn mice. We administered a daily, low oral dose of pentylenetetrazol (PTZ) in milk to Ts65Dn mice for 17 days. Decision-making was tested with and without PTZ treatment. Short and long-term memories were tested before, immediately after, and 1 month following PTZ treatment. Electro-encephalography was also recorded at these three time points to study the effect of the treatment on sleep. We showed that PTZ treatment improved long-term recognition, but not short term memory and led to more Ts65Dn mice showing safer decision-making behavior. PTZ treatment showed a moderate and only global beneficial effect on sleep by decreasing the global amount of wake and increasing non-rapid eye movement sleep in the Ts65Dn mice, which may explain the observed cognitive improvements. These results bring new knowledge on the role of GABA in sleep, memory consolidation, and decision-making abilities in DS.

Down syndrome (DS) is a common genetic condition affecting people worldwide. It involves cognitive disabilities for which there are no drug therapies. The Ts65Dn mouse model of DS shows cognitive impairment due to a reduction in neuron number and connectivity as well as excessive neuronal activity, as [gamma]-aminobutyric acid (GABA) antagonist treatment restores memory in these mice. Our study showed the effects of GABA antagonist treatment on sleep and decision-making in Ts65Dn mice. We administered a daily, low oral dose of pentylenetetrazol (PTZ) in milk to Ts65Dn mice for 17 days. Decision-making was tested with and without PTZ treatment. Short and long-term memories were tested before, immediately after, and 1 month following PTZ treatment. Electro-encephalography was also recorded at these three time points to study the effect of the treatment on sleep. We showed that PTZ treatment improved long-term recognition, but not short term memory and led to more Ts65Dn mice showing safer decision-making behavior. PTZ treatment showed a moderate and only global beneficial effect on sleep by decreasing the global amount of wake and increasing non-rapid eye movement sleep in the Ts65Dn mice, which may explain the observed cognitive improvements. These results bring new knowledge on the role of GABA in sleep, memory consolidation, and decision-making abilities in DS. Key words: cognitive function; learning and memory; sleep/wake cognition; EEG analysis; neuropharmacology

The rise of antimicrobial resistant pathogens can be attributed to the lack of a rapid pathogen identification (ID) or antimicrobial susceptibility testing (AST), resulting in delayed therapeutic decision at the point of care. Gonorrhea is usually empirically treated with no AST results available before treatment, thus contributing to rapid rise in drug resistance. Herein we present a rapid AST platform using RNA signatures for Neisseria gonorrhoeae (NG). RNA-seq followed by bioinformatic tools were applied to explore potential markers in transcriptome profile of NG upon minutes of azithromycin exposure. Validation of candidate markers using PCR showed that two markers (arsR (NGO1562) and rpsO) can deliver accurate AST results across 14 tested isolates. Further validation of our cutoff in comparison to MIC across 64 more isolates confirmed reliability of our platform. Our RNA markers combined with emerging molecular point-of-care systems has the potential to greatly accelerate both ID and AST to inform treatment. Competing Interest Statement The authors have declared no competing interest.

Pump–probe experiments with subfemtosecond resolution are the key to understanding electronic dynamics in quantum systems. Here we demonstrate the generation and control of subfemtosecond pulse pairs from a two-colour X-ray free-electron laser. By measuring the delay between the two pulses with an angular streaking diagnostic, we characterize the group velocity of the X-ray free-electron laser and show control of the pulse delay down to 270 as. We confirm the application of this technique to a pump–probe measurement in core-ionized para -aminophenol. These results reveal the ability to perform pump–probe experiments with subfemtosecond resolution and atomic site specificity. Researchers have demonstrated the generation and control of subfemtosecond pulse pairs from a two-colour X-ray free-electron laser and conducted pump–probe experiments in core-ionized molecules.

Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform (“Artificial Targets”) to characterize CAR T cell function in vitro . These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.

Freshwater ecosystems are highly biodiverse1 and important for livelihoods and economic development2, but are under substantial stress3. To date, comprehensive global assessments of extinction risk have not included any speciose groups primarily living in freshwaters. Consequently, data from predominantly terrestrial tetrapods4,5 are used to guide environmental policy6 and conservation prioritization7, whereas recent proposals for target setting in freshwaters use abiotic factors8–13. However, there is evidence14–17 that such data are insufficient to represent the needs of freshwater species and achieve biodiversity goals18,19. Here we present the results of a multi-taxon global freshwater fauna assessment for The IUCN Red List of Threatened Species covering 23,496 decapod crustaceans, fishes and odonates, finding that one-quarter are threatened with extinction. Prevalent threats include pollution, dams and water extraction, agriculture and invasive species, with overharvesting also driving extinctions. We also examined the degree of surrogacy of both threatened tetrapods and freshwater abiotic factors (water stress and nitrogen) for threatened freshwater species. Threatened tetrapods are good surrogates when prioritizing sites to maximize rarity-weighted richness, but poorer when prioritizing based on the most range-restricted species. However, they are much better surrogates than abiotic factors, which perform worse than random. Thus, although global priority regions identified for tetrapod conservation are broadly reflective of those for freshwater faunas, given differences in key threats and habitats, meeting the needs of tetrapods cannot be assumed sufficient to conserve freshwater species at local scales.

Changes in the intestinal microbiome and microbiota-derived metabolites predict clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report that desaminotyrosine (DAT), a product of bacterial flavonoid metabolism, correlates with improved overall survival and reduced relapse rates in patients receiving allo-HSCT. In preclinical mouse models, treatment with synthetic DAT prevents graft-versus-host disease by protecting the intestinal barrier and promoting intestinal regeneration and contributes to graft-vs.-leukemia responses. DAT´s beneficial effects on intestinal regeneration remain effective despite broad-spectrum antibiotics-induced dysbiosis, also when administered by fecal microbiota transfer with flavonoid-degrading F. plautii . Mechanistically, DAT promotes mTORC1-dependent activation and proliferation of intestinal stem cells, with concomitant engagement of the innate immune receptor STING required to mitigate metabolic stress and maintain an undifferentiated stem cell state independently of type-I interferon responses. Additionally, DAT can skew T cells towards an effector phenotype to modulate graft-versus-leukemia responses. Our data uncover DAT’s dual, tissue- and immune-modulating properties and underscore its potential in precision microbiome-based therapies to improve tissue regeneration and minimize immune-mediated side effects. The success of allogeneic hematopoietic stem cell transplantation for the treatment of haematological cancers is limited by the morbidity and mortality associated with graft-versus-host disease (GVHD). Here the authors show that the microbial metabolite desaminotyrosine contributes to graft-versus-leukemia responses while protecting against GVHD and promoting mTORC1 and STING-dependent intestinal regeneration.