miR-154-5p Is a Novel Endogenous Ligand for TLR7 Inducing Microglial Activation and Neuronal Injury.

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.

Pilot Study of Ondansetron in Improvement of Pediatric Colonoscopy Preparation Outcomes at an Urban Academic Center.

Objectives: To gather initial data on the effectiveness and tolerability of the addition of Ondansetron to bowel preparation regimens to justify a funded, larger, placebo-controlled study. Methods: Design, Setting, and Participants:: A total of 41 pediatric and young adult (age 2-22) patients participated in a single center, open label, parallel randomized trial, with simple randomization. All patients were recruited as outpatients, and all procedures occurred as outpatient procedures, with both recruitment and procedures occurring at a low-resource urban academic medical center in Brooklyn.Interventions and Outcome Measures:: The intervention studied was a single dose of oral-dissolving tablet Ondansetron provided before initiation of bowel preparation using a standardized prep of Polyethylene Glycol 3350 and Bisacodyl. There were 2 arms, a study arm using typical preparation (Polyethylene Glycol 3350 and Bisacodyl) and Ondansetron, and a control arm (Polyethylene Glycol 3350 and Bisacodyl). Patients received standard weight-based dosing. The primary outcome measure assessed was the Boston Bowel Preparation Scale (BBPS) to assess efficacy of preparation. Secondary objectives included evaluation of patient satisfaction via a survey answered by each patient. The questionnaire assessed the presence of the following symptoms during bowel prep: abdominal pain, nausea, bloating, vomiting, scale of ease/difficulty, and if the entire bowel prep was completed. Results: No benefit to BBPS from the addition of Ondansetron to bowel preparation was observed. Statistically significant improvement in reports of abdominal pain (35% decrease in Ondansetron arm) was noted with a P = 0.019. No statistically significant improvement was noted in other symptoms although all domains showed nonsignificant improvement in the Ondansetron arm. Conclusion: No benefit to efficacy of preparation as measured by the BBPS was observed. A single dose of Ondansetron before bowel preparation reduced reports of abdominal pain by 35%, with other symptomatic improvements suggesting possible improvements to be confirmed by a higher-powered study. Trial registration: NCT05439772.

Comparison of Risk and Severity of Helicobacter Pylori Infection in Non-Native Versus US Native Pediatric Patients.

Introduction: Helicobacter pylori (HP) infection is associated with gastritis, peptic ulcer disease (PUD) in the stomach and duodenum, and an increased risk of gastric cancer. The risk of infection, secondary symptoms, and negative outcomes is known to be increased in low- and middle-income countries and vastly less substantial in the United States and Europe. Current North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines recommend endoscopic diagnosis and susceptibility-guided therapy, which is not reflected by current adult guidelines for therapy. In this study, we complete a single-center retrospective review of HP risk by nativity status, as well as the results of the use of standard empiric therapy in HP and PUD patients. Methods: We retrospectively reviewed all endoscopies with patients aged 1-21 years with a known nativity status and identified all HP diagnoses. We also completed the classification of Kyoto scores and classified patients as gastritis versus PUD. Treatment records were obtained, as well as downstream documentation of the impact of empiric therapy. HP prevalence and severity were compared between non-native and native US populations. Results: In total 332 patients were identified, with 59 HP diagnoses. However, 64 patients were immigrants, and 268 were US natives. Totally 39.1% of all immigrant patients had an endoscopically identified HP infection, compared to only 12.7% of US native patients (P < 0.01, relative risk 3.07). HP severity was worse in immigrant patients (Kyoto score 1.5 versus 0.89; P = 0.008). Empiric high-dose amoxicillin triple therapy was equally effective in reducing symptoms in gastritis versus PUD patients. Conclusions: Immigrant patients have a substantially higher risk and severity of HP infection than US natives. Empiric therapy remains highly effective at relieving symptoms. These findings in aggregate suggest that North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines may not adequately serve non-native pediatric patients, with an additional prospective multicenter study needed to confirm. In addition, a prospective study of treatment based on stool antigen tests, as well as a larger prospective study of empiric therapy, may suggest alterations to our approach in line with recent changes to adult Gastroenterology practice.

Epidemiologic Assessment of Pediatric Inflammatory Bowel Disease Presentation in NYC During COVID-19.

Inflammatory bowel disease (IBD) pathogenesis is thought to be induced by a mix of genetic susceptibility, microbial populations, and immune triggers such as infections. Severe acute respiratory syndrome coronavirus 2 (SARS-nCoV2) may have increased capacity to generate autoimmune disease as evidenced by known spikes in diseases such as type 1 diabetes mellitus. Public health interventions like masking and closures additionally created remarkable drops in typical viral infections, with remarkable shifts in influenza-like illness reporting in 2020. This study aims to evaluate the impact of SARS-nCoV2 and associated interventions on pediatric IBD presentation in New York City using records of new diagnoses at a consortium of 4 institutions between 2016 and June 2022. We fit time series model (autoregressive integrated moving average model) to monthly and quarterly number of cases of each disease for January 2016-March 2020 and forecast the period between April 2020 and June 2022. We note no decrease in ulcerative colitis (UC) or Crohn disease (CD) in the aftermath of historic low levels of overall viral illness, and statistically significant increases in CD diagnoses and elevation in UC diagnoses creating a trend suggesting overall increase in IBD diagnoses exceeding the baseline rate of increase. These data suggest a possible linkage between SARS-nCoV2 infection rates and subsequent pediatric IBD presentation.

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation.

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth stunting, and diminished oral vaccine responses. Although EE has been shown to be the by-product of a recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE by performing high-throughput, single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE in Lusaka, Zambia (eight HIV-negative and three HIV-positive), six adults without EE in Boston, United States, and two adults in Durban, South Africa, which we complemented with published data from three additional individuals from the same clinical site. We analyzed previously defined bulk-transcriptomic signatures of reduced villus height and decreased microbial translocation in EE and showed that these signatures may be driven by an increased abundance of surface mucosal cells-a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we determined cell subsets whose fractional abundances associate with EE severity, small intestinal region, and HIV infection. Furthermore, by comparing duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and increased proinflammatory cytokine gene expression in a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells in the EE cohort. Together, our work elucidates epithelial and immune correlates of EE and nominates cellular and molecular targets for intervention.

Elevated Fecal Mitochondrial DNA from Symptomatic Norovirus Infections Suggests Potential Health Relevance of Human Mitochondrial DNA in Fecal Source Tracking.

An end goal of fecal source tracking (FST) is to provide information on risk of transmission of waterborne illnesses associated with fecal contamination. Ideally, concentrations of FST markers in ambient waters would reflect exposure risk. Human mtDNA is an FST marker that is exclusively human in origin and may be elevated in feces of individuals experiencing gastrointestinal inflammation. In this study, we examined whether human mtDNA is elevated in fecal samples from individuals with symptomatic norovirus infections using samples from the United States (US), Mozambique, and Bangladesh. We quantified hCYTB484 (human mtDNA) and HF183/BacR287 (human-associated Bacteroides) FST markers using droplet digital polymerase chain reaction. We observed the greatest difference in concentrations of hCYTB484 when comparing samples from individuals with symptomatic norovirus infections versus individuals without norovirus infections or diarrhea symptoms: log10 increase of 1.42 in US samples (3,820% increase, p-value = 0.062), 0.49 in Mozambique (308% increase, p-value = 0.061), and 0.86 in Bangladesh (648% increase, p-value = 0.035). We did not observe any trends in concentrations of HF183/BacR287 in the same samples. These results suggest concentrations of fecal mtDNA may increase during symptomatic norovirus infection and that mtDNA in environmental samples may represent an unambiguously human source-tracking marker that correlates with enteric pathogen exposure risk.

Improving ondansetron use and oral rehydration instructions for pediatric acute gastroenteritis.

In paediatric patients with acute gastroenteritis (AGE), ondansetron use decreases the need for intravenous fluids, reduces hospitalisations and shortens illness duration. Oral rehydration is also known to have excellent outcomes for mild to moderate dehydration secondary to AGE. Although these interventions are recommended in guidelines from international professional societies, baseline data at our clinic showed that <2% of these patients were offered ondansetron, and that few patients received appropriately detailed rehydration instructions. Therefore, we engaged residents and fellows as teachers and leaders in our university clinic's quality improvement programme to promote evidence-based practice for paediatric AGE. Our gap analysis identified opportunities for interventions including educating paediatricians and paediatrics residents on the safety and utility of the medication. We created standardised oral rehydration after-visit instructions and implemented a trainee-led educational approach that encouraged appropriate medication use. We used a follow-up survey to uncover provider concerns and tailor future interventions. The process metrics included: proportion of paediatric patients appropriately treated with ondansetron (goal of 80%), and proportion of patients given appropriate oral rehydration instructions. The outcome metric was 7-day representation rates. To achieve sustainability, we restructured our process to have senior residents take ownership of teaching and data collection. Trainee-driven interventions increased ondansetron prescription rates to a median of 66.6%. Patients prescribed ondansetron were less likely to represent to care, although representation rate was low overall. Postintervention data suggests that prescription rates decreased without continued interventions and additional systems redesign may help sustain impact.

Histologically Remarkable Eosinophilic Esophagitis Responsive to Dupilumab in a Gastrostomy Tube-Dependent Pediatric Patient on Amino Acid Formula.

Eosinophilic esophagitis (EoE), an eosinophil predominant, TH2-mediated condition increasing in prevalence in pediatric and adult populations, is typically treated with dietary manipulations to avoid triggering antigens. However, identifying specific dietary causes remains a persistent challenge, and restrictive diets are burdensome. Total dietary modification using amino acid-based formula does not always produce symptomatic or histologic resolution, suggesting that exposure to ingested aeroallergens drives their disease. EoE patients demonstrate symptomatic exacerbation from July to September correlating with higher grass and ragweed pollen counts. We present a 7-year-old tracheostomy- and gastrostomy-dependent girl who was found on surveillance endoscopy to have profound eosinophilic infiltration throughout the esophagus with inflammatory changes including basal cell hyperplasia on histology. She responded partially to topical corticosteroid therapy with fluticasone and had complete resolution of esophageal eosinophilic infiltrate with subcutaneous dupilumab.

Persistent SARS-CoV-2 Nucleocapsid Protein Presence in the Intestinal Epithelium of a Pediatric Patient 3 Months After Acute Infection.

In addition to the severe impact of acute respiratory disease during the SARS-CoV-2 pandemic, the issue of "Long COVID" illness has impacted large numbers of patients following the initial infection. Wide ranges of Long Covid incidence have been reported, ranging from 30 to 87%. Long COVID has a variety of clinical manifestations, including gastrointestinal symptoms. Here, we report a case of persistent abdominal pain, 3 months following a SARS-CoV-2 diagnosis, associated with chronic colonic inflammation and the presence of mucosal SARS-CoV-2 virions.

Distinct SARS-CoV-2 RNA fragments activate Toll-like receptors 7 and 8 and induce cytokine release from human macrophages and microglia.

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

The Cutting Edge of Gastroenteritis: Advances in Understanding of Enteric Infection.

ABSTRACT: In recent years, multiple advances have been made in the care, diagnosis, and mechanistic understanding of acute gastroenteritis (AGE). In this review, we discuss the current state of the art of diagnosis and management, as well as how changes in practice can improve care and decrease costs. We will discuss present study demonstrating the effect of AGE on the microbiome and how that may be linked to secondary effects or long-term changes. We will explore the use of novel technologies to further our capacity to understand how gastrointestinal infections occur and promulgate. Finally, will discuss advances in our understanding of how gastrointestinal infections capacitate other changes such as post-viral motility or other post viral intestinal dysfunction.

MicroRNA-100-5p and microRNA-298-5p released from apoptotic cortical neurons are endogenous Toll-like receptor 7/8 ligands that contribute to neurodegeneration.

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.

UNC93B1 Is Widely Expressed in the Murine CNS and Is Required for Neuroinflammation and Neuronal Injury Induced by MicroRNA let-7b.

The chaperone protein Unc-93 homolog B1 (UNC93B1) regulates internalization, trafficking, and stabilization of nucleic acid-sensing Toll-like receptors (TLR) in peripheral immune cells. We sought to determine UNC93B1 expression and its functional relevance in inflammatory and injurious processes in the central nervous system (CNS). We found that UNC93B1 is expressed in various CNS cells including microglia, astrocytes, oligodendrocytes, and neurons, as assessed by PCR, immunocyto-/histochemistry, and flow cytometry. UNC93B1 expression in the murine brain increased during development. Exposure to the microRNA let-7b, a recently discovered endogenous TLR7 activator, but also to TLR3 and TLR4 agonists, led to increased UNC93B1 expression in microglia and neurons. Microglial activation by extracellular let-7b required functional UNC93B1, as assessed by TNF ELISA. Neuronal injury induced by extracellular let-7b was dependent on UNC93B1, as UNC93B1-deficient neurons were unaffected by the microRNA's neurotoxicity in vitro. Intrathecal application of let-7b triggered neurodegeneration in wild-type mice, whereas mice deficient for UNC93B1 were protected against injurious effects on neurons and axons. In summary, our data demonstrate broad UNC93B1 expression in the murine brain and establish this chaperone as a modulator of neuroinflammation and neuronal injury triggered by extracellular microRNA and subsequent induction of TLR signaling.

The circadian clock regulates rhythmic erythropoietin expression in the murine kidney.

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy.

Structure-aware machine learning identifies microRNAs operating as Toll-like receptor 7/8 ligands.

MicroRNAs (miRNAs) can serve as activation signals for membrane receptors, a recently discovered function that is independent of the miRNAs' conventional role in post-transcriptional gene regulation. Here, we introduce a machine learning approach, BrainDead, to identify oligonucleotides that act as ligands for single-stranded RNA-detecting Toll-like receptors (TLR)7/8, thereby triggering an immune response. BrainDead was trained on activation data obtained from in vitro experiments on murine microglia, incorporating sequence and intra-molecular structure, as well as inter-molecular homo-dimerization potential of candidate RNAs. The method was applied to analyse all known human miRNAs regarding their potential to induce TLR7/8 signalling and microglia activation. We validated the predicted functional activity of subsets of high- and low-scoring miRNAs experimentally, of which a selection has been linked to Alzheimer's disease. High agreement between predictions and experiments confirms the robustness and power of BrainDead. The results provide new insight into the mechanisms of how miRNAs act as TLR ligands. Eventually, BrainDead implements a generic machine learning methodology for learning and predicting the functions of short RNAs in any context.

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors.

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.

Identification of CNS Injury-Related microRNAs as Novel Toll-Like Receptor 7/8 Signaling Activators by Small RNA Sequencing.

Toll-like receptors (TLRs) belong to pattern recognition receptors, which respond to danger signals such as pathogen-associated molecular patterns or damage-associated molecular patterns. Upon TLR activation in microglia, the major immune cells in the brain, distinct signaling cascades trigger the production of inflammatory molecules, being a critical feature in neuroinflammation and neurodegenerative processes. Recently, individual microRNAs (miRNAs) were shown to act as endogenous TLR ligands. Here, we conducted systematic screening for miRNAs as potential TLR7/8 ligands by small RNA sequencing of apoptotic neurons and their corresponding supernatants. Several miRNA species were identified in both supernatants and injured neurons, and 83.3% of the media-enriched miRNAs activated murine and/or human TLR7/8 expressed in HEK293-derived TLR reporter cells. Among the detected extracellular miRNAs, distinct miRNAs such as miR-340-3p and miR-132-5p induced cytokine and chemokine release from microglia and triggered neurotoxicity in vitro. Taken together, our systematic study establishes miRNAs released from injured neurons as new TLR7/8 activators, which contribute to inflammatory and neurodegenerative responses in the central nervous system (CNS).

Functional characterization of the circadian clock in the Antarctic krill, Euphausia superba.

Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database "KrillDB" allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment's challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.