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Background Adequate height attainment during early childhood foreshadows lifelong health. Stunting may be caused by environmental enteric dysfunction but has persisted after improvements in water and sanitation access. Recent studies suggest tryptophan and other amino acids improve height attainment. Methods This study used ecological data from 94 countries over 21 years (2000–20) ( n  = 1836) to evaluate the association between the percentage of children with stunting and 4 normalized environmental variables: access to adequate water, sanitation, urban residence, and country-level supply of animal protein. We used mixed effects linear models controlling for year with varying intercepts by country after verifying residuals were uncorrelated and had uniform variance. Results Water and sanitation access, urban residence, and animal protein supply are all associated with lower stunting: 6 percentage-points for each standard deviation (s.d.) change in water, 3 percentage-points for each s.d. change in sanitation, 3 percentage-points for each s.d. change in urban residence, and 1.5 percentage-points for each s.d. change in animal protein, controlling for time. Conclusions Improvements in water and sanitation infrastructure and animal protein access are both important interventions to reduce childhood stunting. These findings complement the 2023 FAO report conclusions regarding the importance of animal protein to childhood growth.

The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period--a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations.

The pandemic coronavirus disease 19 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is marked by thromboembolic events and an inflammatory response throughout the body, including the brain. Employing the machine learning approach BrainDead we systematically screened for SARS-CoV-2 genome-derived single-stranded (ss) RNA fragments with high potential to activate the viral RNA-sensing innate immune receptors Toll-like receptor (TLR)7 and/or TLR8. Analyzing HEK TLR7/8 reporter cells we tested such RNA fragments with respect to their potential to induce activation of human TLR7 and TLR8 and to activate human macrophages, as well as iPSC-derived human microglia, the resident immune cells in the brain. We experimentally validated several sequence-specific RNA fragment candidates out of the SARS-CoV-2 RNA fragments predicted in silico as activators of human TLR7 and TLR8. Moreover, these SARS-CoV-2 ssRNAs induced cytokine release from human macrophages and iPSC-derived human microglia in a sequence- and species-specific fashion. Our findings determine TLR7 and TLR8 as key sensors of SARS-CoV-2-derived ssRNAs and may deepen our understanding of the mechanisms how this virus triggers, but also modulates an inflammatory response through innate immune signaling.

MicroRNAs (miRNAs) canonically regulate post-transcriptional gene expression, but they can also serve as ligands for Toll-like receptors (TLRs). These receptors and their associated signalling pathways contribute to inflammatory responses involved in various central nervous system (CNS) diseases, including Alzheimer’s disease (AD). Here, we investigated the effects of extracellularly delivered miRNA in the context of neuroinflammation. We identified several miRNAs specifically dysregulated in AD and/or neuroinflammatory states, which directly activate the single-stranded RNA sensors mouse TLR7 and human TLR7/8. Among them, extracellular miR-29a-5p induced cytokine and chemokine release from murine primary microglia, altered expression of TLR signalling elements, and enhanced Aβ phagocytosis. Furthermore, this miRNA induced neuronal injury dependent on microglial TLR7 expression, but also in a cell-autonomous fashion, in vitro. Intrathecal injection of miR-29a-5p into mice led to microglial accumulation and neuronal injury in the cerebral cortex through TLR7 after 3 days. Brains of wild-type and APP/PS1 mice, an established AD mouse model, treated with multiple intrathecal miR-29a-5p injections over 120 days exhibited changes in cytokine/chemokine expression and neuronal injury. RNAseq analysis of the cerebral cortex of both miRNA-treated genotypes revealed downregulation of MAPK-associated pathways. Our study establishes AD-associated miRNAs such as miR-29a-5p as TLR7 agonists and signalling molecules for microglia, thereby altering the neuroinflammatory response.

Background MicroRNA (miRNA) expression in the brain is altered in neurodegenerative diseases. Recent studies demonstrated that selected miRNAs conventionally regulating gene expression at the post-transcriptional level can act extracellularly as signaling molecules. The identity of miRNA species serving as membrane receptor ligands involved in neuronal apoptosis in the central nervous system (CNS), as well as the miRNAs’ sequence and structure required for this mode of action remained largely unresolved. Methods Using a microarray-based screening approach we analyzed apoptotic cortical neurons of C56BL/6 mice and their supernatant with respect to alterations in miRNA expression/presence. HEK-Blue Toll-like receptor (TLR) 7/8 reporter cells, primary microglia and macrophages derived from human and mouse were employed to test the potential of the identified miRNAs released from apoptotic neurons to serve as signaling molecules for the RNA-sensing receptors. Biophysical and bioinformatical approaches, as well as immunoassays and sequential microscopy were used to analyze the interaction between candidate miRNA and TLR. Immunocytochemical and -histochemical analyses of murine CNS cultures and adult mice intrathecally injected with miRNAs, respectively, were performed to evaluate the impact of miRNA-induced TLR activation on neuronal survival and microglial activation. Results We identified a specific pattern of miRNAs released from apoptotic cortical neurons that activate TLR7 and/or TLR8, depending on sequence and species. Exposure of microglia and macrophages to certain miRNA classes released from apoptotic neurons resulted in the sequence-specific production of distinct cytokines/chemokines and increased phagocytic activity. Out of those miRNAs miR-100-5p and miR-298-5p, which have consistently been linked to neurodegenerative diseases, entered microglia, located to their endosomes, and directly bound to human TLR8. The miRNA-TLR interaction required novel sequence features, but no specific structure formation of mature miRNA. As a consequence of miR-100-5p- and miR-298-5p-induced TLR activation, cortical neurons underwent cell-autonomous apoptosis. Presence of miR-100-5p and miR-298-5p in cerebrospinal fluid led to neurodegeneration and microglial accumulation in the murine cerebral cortex through TLR7 signaling. Conclusion Our data demonstrate that specific miRNAs are released from apoptotic cortical neurons, serve as endogenous TLR7/8 ligands, and thereby trigger further neuronal apoptosis in the CNS. Our findings underline the recently discovered role of miRNAs as extracellular signaling molecules, particularly in the context of neurodegeneration.

The mammalian circadian clock and the cell cycle are two major biological oscillators whose coupling influences cell fate decisions. In the present study, we use a model-driven experimental approach to investigate the interplay between clock and cell cycle components and the dysregulatory effects of RAS on this coupled system. In particular, we focus on the Ink4a/Arf locus as one of the bridging clock-cell cycle elements. Upon perturbations by the rat sarcoma viral oncogene (RAS), differential effects on the circadian phenotype were observed in wild-type and Ink4a/Arf knock-out mouse embryonic fibroblasts (MEFs), which could be reproduced by our modelling simulations and correlated with opposing cell cycle fate decisions. Interestingly, the observed changes can be attributed to in silico phase shifts in the expression of core-clock elements. A genome-wide analysis revealed a set of differentially expressed genes that form an intricate network with the circadian system with enriched pathways involved in opposing cell cycle phenotypes. In addition, a machine learning approach complemented by cell cycle analysis classified the observed cell cycle fate decisions as dependent on Ink4a/Arf and the oncogene RAS and highlighted a putative fine-tuning role of Bmal1 as an elicitor of such processes, ultimately resulting in increased cell proliferation in the Ink4a/Arf knock-out scenario. This indicates that the dysregulation of the core-clock might work as an enhancer of RAS-mediated regulation of the cell cycle. Our combined in silico and in vitro approach highlights the important role of the circadian clock as an Ink4a/Arf-dependent modulator of oncogene-induced cell fate decisions, reinforcing its function as a tumour-suppressor and the close interplay between the clock and the cell cycle network.

Toll-like receptors (TLRs) are a collection of pattern recognition sensors that form a first line of defence by detecting pathogen- or damage-associated molecular patterns and initiating an inflammatory response. TLR activation in microglia, the major immune cells in the brain, can trigger the release of inflammatory molecules, which may contribute to various CNS diseases including Alzheimer’s disease. Recently, some microRNAs were shown to serve as signalling molecules for TLRs. Here, we present miR-154-5p as a novel TLR7 ligand. Exposing microglia to miR-154-5p results in cytokine release and alters expression of the TLR signalling pathway dependent on TLR7. Additionally, miR-154-5p causes neuronal injury in enriched cortical neuron cultures and additive toxicity in the presence of microglia. Finally, intrathecal injection of miR-154-5p into mice leads to neuronal injury and accumulation of microglia in the cerebral cortex dependent on TLR7 expression. In conclusion, this study establishes miR-154-5p as a direct activator of TLR7 that can cause neuroinflammation and neuronal injury, which may contribute to CNS disease.

MicroRNAs (miRNAs) are abundantly expressed in the brain and are specifically dysregulated in central nervous system (CNS) diseases. They act as post-transcriptional gene regulators but can also serve as ligands for Toll-like receptors (TLRs). This study aims to investigate CNS disease-associated miRNAs as signaling molecules for human microglia. Using a machine learning algorithm and the disease-linked database PhenoMiR, we identified Alzheimer's disease (AD)- and glioma-associated miRNAs as ligands for TLR7 and TLR8. Expression of human TLR7 and TLR8 in iPSC-derived human microglia-like cells (iMGLs) was validated by RT-qPCR. Using ELISA, scratch assay, and FACS, we investigated the miRNAs' potential to modulate iMGL function, including cytokine release, motility, and phagocytosis, respectively. The selective human TLR8 antagonist CU-CPT9a was used to determine the role of this receptor in miRNA-induced modulation of human microglial activity. Co-cultures of iMGLs and iPSC-derived human cortical neurons (iNeurons) were analyzed by Neurotrack imaging to assess the effects of miRNAs on human neurites. We identified AD- and glioma-associated miR-9-5p, miR-132-5p, miR-340-3p, miR-30e-3p, miR-501-3p, and as ligands for human TLR7 and TLR8. Exposure of iMGLs to select miRNAs, including miR-9-5p, miR-132-5p, and miR-340-3p, led to interleukin-6 (IL-6) and tumor necrosis factor (TNF) mRNA expression and protein release in a sequence-dependent fashion. Also, these miRNAs acting as signaling molecules, modulated iMGL motility and phagocytosis activity. The miRNA-induced effects on iMGLs were abolished by CU-CPT9a. Extracellular delivery of miR-132-5p and miR-9-5p to co-cultures of iNeurons and iMGLs resulted in reduced neurite length. Our data establish that distinct CNS disease-associated miRNAs serve as signaling molecules for human microglia via TLR8, thereby controlling the diverse microglial functions and modulating the neuroinflammatory response.

Genetic and biochemical assays reveal that carbon monoxide produced by heme catabolism influences circadian rhythm in mammals by altering the activity of transcription factor CLOCK–BMAL1 at clock-gene targets. Circadian clocks are cell-autonomous oscillators regulating daily rhythms in a wide range of physiological, metabolic and behavioral processes. Feedback of metabolic signals, such as redox state, NAD + /NADH and AMP/ADP ratios, or heme, modulate circadian rhythms and thereby optimize energy utilization across the 24-h cycle. We show that rhythmic heme degradation, which generates the signaling molecule carbon monoxide (CO), is required for normal circadian rhythms as well as circadian metabolic outputs. CO suppresses circadian transcription by attenuating CLOCK–BMAL1 binding to target promoters. Pharmacological inhibition or genetic depletion of CO-producing heme oxygenases abrogates normal daily cycles in mammalian cells and Drosophila . In mouse hepatocytes, suppression of CO production leads to a global upregulation of CLOCK–BMAL1-dependent circadian gene expression and dysregulated glucose metabolism. Together, our findings show that CO metabolism is an important link between the basic circadian-clock machinery, metabolism and behavior.

Background Dysregulation of microRNA (miRNA) expression in the brain is a common feature of neurodegenerative diseases. Beyond their conventional role in regulating gene expression at the post-transcriptional level, certain miRNAs can act extracellularly as signaling molecules. Our study elucidates the identity of such miRNA species serving as ligands for membrane receptors expressed in central nervous system (CNS) neurons and the impact of such miRNAs on neurons in the context of neurodegenerative disease. Methods We combined a machine learning approach with the analysis of disease-associated miRNA databases to predict Alzheimer’s disease (AD)-associated miRNAs as potential signaling molecules for single-stranded RNA-sensing Toll-like receptors (TLRs) 7 and 8. TLR-expressing HEK-Blue reporter cells, primary murine microglia, and human THP-1 macrophages were used to validate the AD miRNAs as ligands for human and mouse TLR7 and/or TLR8. Interaction between mouse cortical neurons and extracellularly applied AD miRNAs was analyzed by live cell imaging and confocal microscopy. Transcriptome changes in cortical neurons exposed to AD miRNAs were assessed by RNAseq and RT-qPCR. The extracellular AD miRNAs’ effects on CNS neuron structure were investigated in cell cultures of murine primary cortical neurons and iPSC-derived human cortical neurons by immunocytochemistry. We employed a mouse model of intrathecal injection to assess effects of AD miRNAs acting as signaling molecules on neurons in vivo. Results We identified the AD-associated miRNAs miR-124-5p, miR-92a-1-5p, miR-9-5p, and miR-501-3p as novel endogenous ligands for TLR7 and/or TLR8. These miRNAs being extracellularly stable and active were taken up by murine cortical neurons via endocytosis and induced changes in neuronal inflammation-, proliferation-, and apoptosis-related gene expression. Exposure of both murine and human cortical neurons to the AD-associated miRNAs led to alterations of dendrite and axon structure, synapse protein expression, and cell viability in a sequence-dependent fashion. Extracellular introduction of the AD miRNAs into the cerebrospinal fluid of mice resulted in both changes in neuronal structure and synapses, and neuronal loss in the cerebral cortex. Most of the observed extracellular miRNA-induced effects on cortical neurons involved TLR7/8 signaling. Conclusion Neurodegenerative disease-associated miRNAs in extracellular form act as signaling molecules for CNS neurons including human cortical neurons, thereby modulating their structure and viability.