Transcription factor Tox2 is required for metabolic adaptation and tissue residency of ILC3 in the gut.

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

TRF2-mediated telomere protection is dispensable in pluripotent stem cells.

In mammals, telomere protection is mediated by the essential protein TRF2, which binds chromosome ends and ensures genome integrity1,2. TRF2 depletion results in end-to-end chromosome fusions in all cell types that have been tested so far. Here we find that TRF2 is dispensable for the proliferation and survival of mouse embryonic stem (ES) cells. Trf2-/- (also known as Terf2) ES cells do not exhibit telomere fusions and can be expanded indefinitely. In response to the deletion of TRF2, ES cells exhibit a muted DNA damage response that is characterized by the recruitment of γH2AX-but not 53BP1-to telomeres. To define the mechanisms that control this unique DNA damage response in ES cells, we performed a CRISPR-Cas9-knockout screen. We found a strong dependency of TRF2-null ES cells on the telomere-associated protein POT1B and on the chromatin remodelling factor BRD2. Co-depletion of POT1B or BRD2 with TRF2 restores a canonical DNA damage response at telomeres, resulting in frequent telomere fusions. We found that TRF2 depletion in ES cells activates a totipotent-like two-cell-stage transcriptional program that includes high levels of ZSCAN4. We show that the upregulation of ZSCAN4 contributes to telomere protection in the absence of TRF2. Together, our results uncover a unique response to telomere deprotection during early development.

ERF deletion rescues RAS deficiency in mouse embryonic stem cells.

MEK inhibition in combination with a glycogen synthase kinase-3ß (GSK3ß) inhibitor, referred as the 2i condition, favors pluripotency in embryonic stem cells (ESCs). However, the mechanisms by which the 2i condition limits ESC differentiation and whether RAS proteins are involved in this phenomenon remain poorly understood. Here we show that RAS nullyzygosity reduces the growth of mouse ESCs (mESCs) and prohibits their differentiation. Upon RAS deficiency or MEK inhibition, ERF (E twenty-six 2 [Ets2]-repressive factor), a transcriptional repressor from the ETS domain family, translocates to the nucleus, where it binds to the enhancers of pluripotency factors and key RAS targets. Remarkably, deletion of Erf rescues the proliferative defects of RAS-devoid mESCs and restores their capacity to differentiate. Furthermore, we show that Erf loss enables the development of RAS nullyzygous teratomas. In summary, this work reveals an essential role for RAS proteins in pluripotency and identifies ERF as a key mediator of the response to RAS/MEK/ERK inhibition in mESCs.

Transition from totipotency to pluripotency in mice: insights into molecular mechanisms.

Totipotency is the ability of a single cell to develop into a full organism and, in mammals, is strictly associated with the early stages of development following fertilization. This unlimited developmental potential becomes quickly restricted as embryonic cells transition into a pluripotent state. The loss of totipotency seems a consequence of the zygotic genome activation (ZGA), a process that determines the switch from maternal to embryonic transcription, which in mice takes place following the first cleavage. ZGA confers to the totipotent cell a transient transcriptional profile characterized by the expression of stage-specific genes and a set of transposable elements that prepares the embryo for subsequent development. The timely silencing of this transcriptional program during the exit from totipotency is required to ensure proper development. Importantly, the molecular mechanisms regulating the transition from totipotency to pluripotency have remained elusive due to the scarcity of embryonic material. However, the development of new in vitro totipotent-like models together with advances in low-input genome-wide technologies, are providing a better mechanistic understanding of how this important transition is achieved. This review summarizes the current knowledge on the molecular determinants that regulate the exit from totipotency.

A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors.

One recurring theme in drug development is to exploit synthetic lethal properties as means to preferentially damage the DNA of cancer cells. We and others have previously developed inhibitors of the ATR kinase, shown to be particularly genotoxic for cells expressing certain oncogenes. In contrast, the mechanisms of resistance to ATR inhibitors remain unexplored. We report here on a genome-wide CRISPR-Cas9 screen that identified CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A-deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A-deficient cells. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells and reveals genetic interactions that could be used for their rational use.

Genome architecture and totipotency: An intertwined relation during early embryonic development.

Chromosomes are not randomly packed and positioned into the nucleus but folded in higher-order chromatin structures with defined functions. However, the genome of a fertilized embryo undergoes a dramatic epigenetic reprogramming characterized by extensive chromatin relaxation and the lack of a defined three-dimensional structure. This reprogramming is followed by a slow genome refolding that gradually strengthens the chromatin architecture during preimplantation development. Interestingly, genome refolding during early development coincides with a progressive loss of developmental potential suggesting a link between chromatin organization and cell plasticity. In agreement, loss of chromatin architecture upon depletion of the insulator transcription factor CTCF in embryonic stem cells led to the upregulation of the transcriptional program found in totipotent cells of the embryo, those with the highest developmental potential. This essay will discuss the impact of genome folding in controlling the expression of transcriptional programs involved in early development and their plastic-associated features.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

Microbiota-Derived Extracellular Vesicles as a Postbiotic Strategy to Alleviate Diarrhea and Enhance Immunity in Rotavirus-Infected Neonatal Rats.

Rotavirus (RV) infection is a major cause of acute gastroenteritis in children under 5 years old, resulting in elevated mortality rates in low-income countries. The efficacy of anti-RV vaccines is limited in underdeveloped countries, emphasizing the need for novel strategies to boost immunity and alleviate RV-induced diarrhea. This study explores the effectiveness of interventions involving extracellular vesicles (EVs) from probiotic and commensal E. coli in mitigating diarrhea and enhancing immunity in a preclinical model of RV infection in suckling rats. On days 8 and 16 of life, variables related to humoral and cellular immunity and intestinal function/architecture were assessed. Both interventions enhanced humoral (serum immunoglobulins) and cellular (splenic natural killer (NK), cytotoxic T (Tc) and positive T-cell receptor γδ (TCRγδ) cells) immunity against viral infections and downregulated the intestinal serotonin receptor-3 (HTR3). However, certain effects were strain-specific. EcoR12 EVs activated intestinal CD68, TLR2 and IL-12 expression, whereas EcN EVs improved intestinal maturation, barrier properties (goblet cell numbers/mucin 2 expression) and absorptive function (villus length). In conclusion, interventions involving probiotic/microbiota EVs may serve as a safe postbiotic strategy to improve clinical symptoms and immune responses during RV infection in the neonatal period. Furthermore, they could be used as adjuvants to enhance the immunogenicity and efficacy of anti-RV vaccines.

Author Correction: Conversion of human fibroblasts to angioblast-like progenitor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbß3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

The maternal side of Fanconi Anemia.

Fanconi anemia is characterized by a higher sensitivity to DNA crosslinking agents, including aldehydes. In this issue of Molecular Cell, Oberbeck et al. (2014) reveal that detoxification of aldehydes by pregnant mothers contributes to limit the severity of the disease.

Human Platelets Contain, Translate, and Secrete Azurocidin; A Novel Effect on Hemostasis.

Platelets play a significant role in hemostasis and perform essential immune functions, evidenced by the extensive repertoire of antimicrobial molecules. Currently, there is no clear description of the presence of azurocidin in human platelets. Azurocidin is a 37 kDa cationic protein abundant in neutrophils, with microbicidal, opsonizing, and vascular permeability-inducing activity. Therefore, this work aimed to characterize the content, secretion, translation, and functions of azurocidin in platelets. Our results show the presence of azurocidin mRNA and protein in α-granules of platelet and megakaryoblasts, and stimulation with thrombin, ADP, and LPS leads to the secretion of free azurocidin as well as within extracellular vesicles. In addition, platelets can translate azurocidin in a basal or thrombin-induced manner. Finally, we found that the addition of low concentrations of azurocidin prevents platelet aggregation and activation. In conclusion, we demonstrate that platelets contain, secrete, and translate azurocidin, and this protein may have important implications for hemostasis.

The cross-sectional association of frailty with past and current exposure to strong anticholinergic drugs.

BACKGROUND: Anticholinergic drugs may contribute to frailty by impairing cognitive and physical functions. Strong anticholinergic drugs in particular may have adverse effects among older adults. OBJECTIVES: Determine the association between frailty and the use of strong anticholinergic drugs among older US Veterans. METHODS: This is a cross-sectional study of community-dwelling Veterans 65 years and older who had determinations of frailty status. Prescription data for patients using strong anticholinergic medications (never/past/current) was obtained via electronic health records. A 31-item VA Frailty Index (VA-FI) was generated at the time of the assessment. We dichotomized the groups into non-frail (FI = < 0.21) and frail (FI ≥ 0.21) patients. We used binomial logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Frailty was the dependent variable and use of strong anticholinergic drugs was the independent variable. Multivariate adjustment was conducted for age, gender, race, ethnicity, marital status, and BMI. RESULTS: Population sample consisted of 17,084 Veterans who were 71.05% Caucasian, 97.34% male, and with a mean age 75.60 (SD = 8.04) years. Among the population, 9940 (58.18%) patients had no previous use of strong anticholinergic drugs, whereas 5182 (30.33%) had past exposure and 1962 (11.49%) had current exposure. In binomial logistic regression, individuals with past (OR 3.27, 95% CI 3.03-3.54, p < 0.0005) or current (OR 4.78, 95% CI 4.30-5.31, p < 0.0005) exposure showed a higher association with frailty as compared to individuals who were never exposed. CONCLUSIONS: Past and current use of strong anticholinergic drugs were associated with frailty in older Veterans. These results suggest that screening for frailty in patients with past or current exposure to strong anticholinergic medications may be necessary for proper management.

Automatic Ankle Angle Detection by Integrated RGB and Depth Camera System.

Depth cameras are developing widely. One of their main virtues is that, based on their data and by applying machine learning algorithms and techniques, it is possible to perform body tracking and make an accurate three-dimensional representation of body movement. Specifically, this paper will use the Kinect v2 device, which incorporates a random forest algorithm for 25 joints detection in the human body. However, although Kinect v2 is a powerful tool, there are circumstances in which the device's design does not allow the extraction of such data or the accuracy of the data is low, as is usually the case with foot position. We propose a method of acquiring this data in circumstances where the Kinect v2 device does not recognize the body when only the lower limbs are visible, improving the ankle angle's precision employing projection lines. Using a region-based convolutional neural network (Mask RCNN) for body recognition, raw data extraction for automatic ankle angle measurement has been achieved. All angles have been evaluated by inertial measurement units (IMUs) as gold standard. For the six tests carried out at different fixed distances between 0.5 and 4 m to the Kinect, we have obtained (mean ± SD) a Pearson's coefficient, r = 0.89 ± 0.04, a Spearman's coefficient, ρ = 0.83 ± 0.09, a root mean square error, RMSE = 10.7 ± 2.6 deg and a mean absolute error, MAE = 7.5 ± 1.8 deg. For the walking test, or variable distance test, we have obtained a Pearson's coefficient, r = 0.74, a Spearman's coefficient, ρ = 0.72, an RMSE = 6.4 deg and an MAE = 4.7 deg.

Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets.

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

Safety of propofol sedation administered by an endoscopy team for outpatient colonoscopy.

Sedation during colonoscopy increases comfort and reduces unexpected patient movement. Colorectal cancer (CRC) screening colonoscopies performed under propofol sedation controlled by our endoscopy team (ET) were retrospectively studied for eight months.

[Negative effects of the patients' rights law and neuro-rights bill in Chile]. / Efectos negativos en la investigación y el quehacer médico en Chile de la Ley 20.584 y la Ley de Neuroderechos en discusión: la urgente necesidad de aprender de nuestros errores.

Recently, the Chilean Senate approved the main ideas of a constitutional reform and a Neuro-rights bill. This bill aims to protect people from the potential abusive use of "neuro-technologies". Unfortunately, a literal interpretation of this law can produce severe negative effects both in the development of neuroscience research and medical practice in Chile, interfering with current treatments in countless patients suffering from neuropsychiatric diseases. This fear stems from the observation of the negative effects that recent Chilean legislations have produced, which share with the Neuro-Rights Law the attempt to protect vulnerable populations from potential abuse from certain medical interventions. In fact, Law 20,584 promulgated in 2012, instead of protecting the most vulnerable patients "incapacitated to consent", produced enormous, and even possibly irreversible, damage to research in Chile in pathologies that require urgent attention, such as many neuropsychiatric diseases. This article details the effects that Law 20.584 had on research in Chile, how it relates to the Neuro-Rights Law, and the potential negative effects that the latter could have on research and medical practice, if it is not formulated correcting its errors.

Involvement of top-down networks in the perception of facial emotions: A magnetoencephalographic investigation.

Conscious perception of the emotional valence of faces has been proposed to involve top-down and bottom-up information processing. Yet, the underlying neuronal mechanisms of these two processes and the implementation of their cooperation is still unclear. According to the global workspace model, higher level cognitive processing of visual emotional stimuli relies on both bottom-up and top-down processing. Using masking stimuli in a visual backward masking paradigm with delays at the perceptual threshold, at which stimuli can only partly be detected, suggests that only top-down processing differs between correctly and incorrectly perceived stimuli, while bottom-up visual processing is not compromised and comparable for both conditions. Providing visual stimulation near the perceptual threshold in the backward masking paradigm thus enabled us to compare differences in top-down modulation of the visual information of correctly and incorrectly recognized facial emotions in 12 healthy individuals using magnetoencephalography (MEG). For correctly recognized facial emotions, we found a right-hemispheric fronto-parietal network oscillating in the high-beta and low-gamma band and exerting top-down control as determined by the causality measure of phase slope index (PSI). In contrast, incorrect recognition was associated with enhanced coupling in the gamma band between left frontal and right parietal regions. Our results indicate that the perception of emotional face stimuli relies on the right-hemispheric dominance of synchronized fronto-parietal gamma-band activity.

Vitamin D deficiency downregulates TASK-1 channels and induces pulmonary vascular dysfunction.

Vitamin D (VitD) receptor regulates the expression of several genes involved in signaling pathways affected in pulmonary hypertension (PH). VitD deficiency is highly prevalent in PH, and low levels are associated with poor prognosis. We investigated if VitD deficiency may predispose to or exacerbate PH. Male Wistar rats were fed with a standard or a VitD-free diet for 5 wk. Next, rats were further divided into controls or PH, which was induced by a single dose of Su-5416 (20 mg/kg) and exposure to hypoxia (10% O2) for 2 wk. VitD deficiency had no effect on pulmonary pressure in normoxic rats, indicating that, by itself, it does not trigger PH. However, it induced several moderate but significant changes characteristic of PH in the pulmonary arteries, such as increased muscularization, endothelial dysfunction, increased survivin, and reduced bone morphogenetic protein (Bmp) 4, Bmp6, DNA damage-inducible transcript 4, and K+ two-pore domain channel subfamily K member 3 (Kcnk3) expression. Myocytes isolated from pulmonary arteries from VitD-deficient rats had a reduced whole voltage-dependent potassium current density and acid-sensitive (TASK-like) potassium currents. In rats with PH induced by Su-5416 plus hypoxia, VitD-free diet induced a modest increase in pulmonary pressure, worsened endothelial function, increased the hyperreactivity to serotonin, arterial muscularization, decreased total and TASK-1 potassium currents, and further depolarized the pulmonary artery smooth muscle cell membrane. In human pulmonary artery smooth muscle cells from controls and patients with PH, the active form of VitD calcitriol significantly increased KCNK3 mRNA expression. Altogether, these data strongly suggest that the deficit in VitD induces pulmonary vascular dysfunction.

Human platelets and megakaryocytes express defensin alpha 1.

Platelets are anucleate cells that have a role in several innate immune functions, including the secretion of proteins with antimicrobial activity. Several studies have demonstrated the ability of platelets to secrete thrombin-induced platelet microbicidal proteins and antimicrobial peptides, like hBD-1. However, the expression and secretion of defensins of the alpha family by platelets have not been fully elucidated. The aim of this study was to characterize the expression of defensin alpha 1 (DEFA1) in human platelets and megakaryocytes. Our data indicate that DEFA1 mRNA and protein are present in peripheral blood platelets and in the megakaryoblastic leukemia cell line (MEG-01). DEFA1 co-localize with α-granules of platelets and MEG-01 cells, and was also detected in cytoplasm of MEG-01 cells. The assay of our in vitro model of platelet-like particles (PLPs) revealed that MEG-01 cells could transfer DEFA1 mRNA to their differentiated PLPs. Furthermore, platelets secreted DEFA1 into the culture medium when activated with thrombin, adenosine diphosphate, and lipopolysaccharide; meanwhile, MEG-01 cells secreted DEFA1 when activated with thrombopoietin. Platelet's secreted DEFA1 can rebind to platelet's surface and have antibacterial activity against the gram-negative bacteria Escherichia coli. In summary, our data indicate that both, human platelets and megakaryocytes, can express and secrete DEFA1. These results suggest a new role of platelets and megakaryocytes in the innate immune response.