Piwi Is Required during Drosophila Embryogenesis to License Dual-Strand piRNA Clusters for Transposon Repression in Adult Ovaries.

Most piRNAs in the Drosophila female germline are transcribed from heterochromatic regions called dual-strand piRNA clusters. Histone 3 lysine 9 trimethylation (H3K9me3) is required for licensing piRNA production by these clusters. However, it is unclear when and how they acquire this permissive heterochromatic state. Here, we show that transient Piwi depletion in Drosophila embryos results in H3K9me3 decrease at piRNA clusters in ovaries. This is accompanied by impaired biogenesis of ovarian piRNAs, accumulation of transposable element transcripts, and female sterility. Conversely, Piwi depletion at later developmental stages does not disturb piRNA cluster licensing. These results indicate that the identity of piRNA clusters is epigenetically acquired in a Piwi-dependent manner during embryonic development, which is reminiscent of the widespread genome reprogramming occurring during early mammalian zygotic development.

Noncommunicable diseases in the Americas: a review of the Pan American Health Organization's 25-year program of work.

This article describes progress in tackling noncommunicable diseases (NCDs) in the Americas since the Pan American Health Organization (PAHO) started its NCD program 25 years ago. Changes in the epidemiology of NCDs, NCD policies, health service capacity, and surveillance are discussed. PAHO's NCD program is guided by regional plans of action on specific NCDs and risk factors, as well as a comprehensive NCD plan. Its work involves implementing evidence-based World Health Organization technical packages on NCDs and their risk factors with the aim of achieving the Sustainable Development Goal target of a one third reduction in premature mortality caused by NCDs by 2030. Important advances have been made in the past 25 years in implementation of: policies on NCD risk factors; interventions to improve NCD diagnosis and treatment; and NCD surveillance. Premature mortality from NCDs decreased by 1.7% a year between 2000 and 2011 and 0.77% a year between 2011 and 2019. However, policies on risk factor prevention and health promotion need to be strengthened to ensure more countries are on track to achieving the NCD-related health goals of the Sustainable Development Goals by 2030. Actions are recommended for governments to raise the priority of NCDs by: making NCDs a core pillar of primary care services, using revenues from health taxes to invest more in NCD prevention and control; and implementing policies, laws, and regulations to reduce the demand for and availability of tobacco, alcohol, and ultra-processed food products.

En este artículo se describe el progreso en la lucha contra las enfermedades no transmisibles (ENT) en la Región de las Américas desde que la Organización Panamericana de la Salud (OPS) iniciara su programa contra las ENT hace 25 años. Se abordan los cambios en las características epidemiológicas, las políticas, la capacidad de los servicios de salud y la vigilancia de estas enfermedades. Este programa de la OPS se rige por planes regionales de acción sobre enfermedades y factores de riesgo específicos, así como por un plan integral de ENT. Su labor consiste en poner en práctica paquetes técnicos de la Organización Mundial de la Salud basados en la evidencia sobre las ENT y sus factores de riesgo con el objetivo de alcanzar la meta de los Objetivos de Desarrollo Sostenible (ODS) de reducir en un tercio la mortalidad prematura causada por las ENT para el 2030. En los últimos 25 años se han logrado importantes avances en la ejecución de políticas sobre los factores de riesgo de estas enfermedades, en las intervenciones para mejorar su diagnóstico y tratamiento, y en la vigilancia. La mortalidad prematura por ENT disminuyó 1,7% anual entre el 2000 y el 2011 y 0,77% anual entre los años 2011 y 2019. Sin embargo, es necesario fortalecer las políticas de prevención de factores de riesgo y promoción de la salud para garantizar que más países estén bien encaminados para lograr las metas de salud de los ODS relacionadas con las ENT para el 2030. Se recomiendan medidas para que los gobiernos prioricen más las ENT y las conviertan en un pilar central de los servicios de atención primaria, al usar los ingresos generados por los impuestos en el sector de la salud para incrementar las inversiones en la prevención y control de las ENT, y ejecutar políticas, leyes y regulaciones para reducir la demanda y la disponibilidad de tabaco, alcohol y alimentos ultraprocesados.

Este artigo descreve o progresso no combate às doenças não transmissíveis (DNTs) nas Américas desde que a Organização Pan-Americana da Saúde (OPAS) iniciou seu programa para essas doenças há 25 anos. Discute-se como evoluíram a epidemiologia das DNTs, as políticas contra essas doenças, a capacidade dos serviços de saúde e a vigilância. O programa da OPAS para as DNTs é orientado por planos de ação regionais sobre DNTs específicas e fatores de risco, bem como por um plano integral contra essas doenças. O trabalho envolve a implementação de pacotes técnicos da Organização Mundial da Saúde baseados em evidências sobre as DNTs e seus fatores de risco, no intuito de alcançar a meta do Objetivo de Desenvolvimento Sustentável de reduzir em um terço a mortalidade prematura causada pelas DNTs até 2030. Avanços importantes foram obtidos nos últimos 25 anos na implementação de políticas sobre fatores de risco das DNTs, intervenções para melhorar o diagnóstico e o tratamento das DNTs, e vigilância das DNTs. A mortalidade prematura causada pelas DNTs diminuiu 1,7% ao ano entre 2000 e 2011 e 0,77% ao ano entre 2011 e 2019. Contudo, as políticas sobre a prevenção dos fatores de risco e a promoção da saúde precisam ser fortalecidas para que mais países estejam no rumo certo para alcançar as metas de saúde relacionadas a essas doenças, no âmbito dos Objetivos de Desenvolvimento Sustentável até 2030. São recomendadas medidas para que os governos elevem a prioridade das DNTs ao torná-las um pilar central dos serviços de atenção primária, usando a receita dos tributos saudáveis para investir mais na prevenção e no controle das DNTs, e ao implementar políticas, leis e regulamentos para reduzir a demanda e a disponibilidade de álcool, tabaco e produtos alimentícios ultraprocessados.

Antarctic desert soil bacteria exhibit high novel natural product potential, evaluated through long-read genome sequencing and comparative genomics.

Actinobacteria and Proteobacteria are important producers of bioactive natural products (NP), and these phyla dominate in the arid soils of Antarctica, where metabolic adaptations influence survival under harsh conditions. Biosynthetic gene clusters (BGCs) which encode NPs, are typically long and repetitious high G + C regions difficult to sequence with short-read technologies. We sequenced 17 Antarctic soil bacteria from multi-genome libraries, employing the long-read PacBio platform, to optimize capture of BGCs and to facilitate a comprehensive analysis of their NP capacity. We report 13 complete bacterial genomes of high quality and contiguity, representing 10 different cold-adapted genera including novel species. Antarctic BGCs exhibited low similarity to known compound BGCs (av. 31%), with an abundance of terpene, non-ribosomal peptide and polyketide-encoding clusters. Comparative genome analysis was used to map BGC variation between closely related strains from geographically distant environments. Results showed the greatest biosynthetic differences to be in a psychrotolerant Streptomyces strain, as well as a rare Actinobacteria genus, Kribbella, while two other Streptomyces spp. were surprisingly similar to known genomes. Streptomyces and Kribbella BGCs were predicted to encode antitumour, antifungal, antibacterial and biosurfactant-like compounds, and the synthesis of NPs with antibacterial, antifungal and surfactant properties was confirmed through bioactivity assays.

Dysregulation of endocannabinoid concentrations in human subcutaneous adipose tissue in obesity and modulation by omega-3 polyunsaturated fatty acids.

Obesity is believed to be associated with a dysregulated endocannabinoid system which may reflect enhanced inflammation. However, reports of this in human white adipose tissue (WAT) are limited and inconclusive. Marine long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have anti-inflammatory actions and therefore may improve obesity-associated adipose tissue inflammation. Therefore, fatty acid (FA) concentrations, endocannabinoid concentrations, and gene expression were assessed in subcutaneous WAT (scWAT) biopsies from healthy normal weight individuals (BMI 18.5-25 kg/m2) and individuals living with metabolically healthy obesity (BMI 30-40 kg/m2) prior to and following a 12-week intervention with 3 g fish oil/day (1.1 g eicosapentaenoic acid (EPA) + 0.8 g DHA) or 3 g corn oil/day (placebo). WAT from individuals living with metabolically healthy obesity had higher n-6 PUFAs and EPA, higher concentrations of two endocannabinoids (anandamide (AEA) and eicosapentaenoyl ethanolamide (EPEA)), higher expression of phospholipase A2 Group IID (PLA2G2D) and phospholipase A2 Group IVA (PLA2G4A), and lower expression of CNR1. In response to fish oil intervention, WAT EPA increased to a similar extent in both BMI groups, and WAT DHA increased by a greater extent in normal weight individuals. WAT EPEA and docosahexaenoyl ethanolamide (DHEA) increased in normal weight individuals only and WAT 2-arachidonyl glycerol (2-AG) decreased in individuals living with metabolically healthy obesity only. Altered WAT fatty acid, endocannabinoid, and gene expression profiles in metabolically healthy obesity at baseline may be linked. WAT incorporates n-3 PUFAs when their intake is increased which affects the endocannabinoid system; however, effects appear greater in normal weight individuals than in those living with metabolically healthy obesity.

Bacterial intracellularly active toxins: Membrane localisation of the active domain.

Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras-GTPases, certain virulence factors of Gram negative bacteria, Rho-GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G-proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four-helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine.

Differential SLC6A4 methylation: a predictive epigenetic marker of adiposity from birth to adulthood.

BACKGROUND: The early life environment may influence susceptibility to obesity and metabolic disease in later life through epigenetic processes. SLC6A4 is an important mediator of serotonin bioavailability, and has a key role in energy balance. We tested the hypothesis that methylation of the SLC6A4 gene predicts adiposity across the life course. METHODS: DNA methylation at 5 CpGs within the SLC6A4 gene identified from a previous methyl binding domain array was measured by pyrosequencing. We measured DNA methylation in umbilical cord (UC) from children in the Southampton Women's Survey cohort (n = 680), in peripheral blood from adolescents in the Western Australian Pregnancy Cohort Study (n = 812), and in adipose tissue from lean and obese adults from the UK BIOCLAIMS cohort (n = 81). Real-time PCR was performed to assess whether there were corresponding alterations in gene expression in the adipose tissue. RESULTS: Lower UC methylation of CpG5 was associated with higher total fat mass at 4 years (p = 0.031), total fat mass at 6-7 years (p = 0.0001) and % fat mass at 6-7 years (p = 0.004). Lower UC methylation of CpG5 was also associated with higher triceps skinfold thickness at birth (p = 0.013), 6 months (p = 0.038), 12 months (p = 0.062), 2 years (p = 0.0003), 3 years (p = 0.00004) and 6-7 years (p = 0.013). Higher maternal pregnancy weight gain (p = 0.046) and lower parity (p = 0.029) were both associated with lower SLC6A4 CpG5 methylation. In adolescents, lower methylation of CpG5 in peripheral blood was associated with greater concurrent measures of adiposity including BMI (p ≤ 0.001), waist circumference (p = 0.011), subcutaneous fat (p ≤ 0.001) and subscapular, abdominal and suprailiac skinfold thicknesses (p = 0.002, p = 0.008, p = 0.004, respectively). In adipose tissue, methylation of both SLC6A4 CpG5 (p = 0.019) and expression of SLC6A4 (p = 0.008) was lower in obese compared with lean adults. CONCLUSIONS: These data suggest that altered methylation of CpG loci within SLC6A4 may provide a robust marker of adiposity across the life course.

Botulinum neurotoxin type B uses a distinct entry pathway mediated by CDC42 into intestinal cells versus neuronal cells.

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis by inhibiting the release of acetylcholine at the neuromuscular junctions. BoNT type B (BoNT/B) most often induces mild forms of botulism with predominant dysautonomic symptoms. In food borne botulism and botulism by intestinal colonisation such as infant botulism, which are the most frequent naturally acquired forms of botulism, the digestive tract is the main entry route of BoNTs into the organism. We previously showed that BoNT/B translocates through mouse intestinal barrier by an endocytosis-dependent mechanism and subsequently targets neuronal cells, mainly cholinergic neurons, in the intestinal mucosa and musculosa. Here, we investigated the entry pathway of BoNT/B using fluorescent C-terminal domain of the heavy chain (HcB), which is involved in the binding to specific receptor(s) and entry process into target cells. While the combination of gangliosides GD1a /GD1b /GT1b and synaptotagmin I and to a greater extent synaptotagmin II constitutes the functional HcB receptor on NG108-15 neuronal cells, HcB only uses the gangliosides GD1a /GD1b /GT1b to efficiently bind to m-ICcl2 intestinal cells. HcB enters both cell types by a dynamin-dependent endocytosis, which is efficiently prevented by Dynasore, a dynamin inhibitor, and reaches a common early endosomal compartment labeled by early endosome antigen (EEA1). In contrast to neuronal cells, HcB uses a Cdc42-dependent pathway to enter intestinal cells. Then, HcB is transported to late endosomes in neuronal cells, whereas it exploits a nonacidified pathway from apical to basal lateral side of m-ICcl2 cells supporting a transcytotic route in epithelial intestinal cells.

Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall.

Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C-terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis-dependent mechanism within 10-20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B.

Perioperative immunonutrition in patients undergoing liver transplantation: a randomized double-blind trial.

UNLABELLED: Preliminary work suggested that perioperative immunonutrition (IMN) enriched in n-3 fatty acids, arginine, and nucleotides may improve preoperative nutritional status, enhance postoperative recovery, and reduce postoperative infectious complications in patients undergoing liver transplantation (LT). The current study examined these outcomes in a double-blind, randomized, controlled trial. Patients wait-listed for LT (n = 120) were randomized to either supplemental (0.6 L/d) oral IMN or an isocaloric control (CON). Enteral IMN or CON was resumed postoperatively and continued for at least 5 days. The change in total body protein (TBP) measured by neutron activation from study entry until immediately prior to LT was the primary endpoint and TBP measurements were repeated 10, 30, 90, 180, and 360 days after LT. Infectious complications were recorded for the first 30 postoperative days. Nineteen patients died or were delisted prior to LT. Fifty-two IMN and 49 CON patients received supplemental nutrition for a median (range) 56 (0-480) and 65 (0-348) days, respectively. Preoperative changes in TBP were not significant (IMN: 0.06 ± 0.15 [SEM]; CON: 0.12 ± 0.10 kg). Compared to baseline, a 0.7 ± 0.2 kg loss of TBP was seen in both groups at 30 days after LT (P < 0.0001) and, at 360 days, TBP had not increased significantly (IMN: 0.08 ± 0.19 kg; CON: 0.26 ± 0.23 kg). Infectious complications occurred in 31 (60%) IMN and 28 (57%) CON patients (P = 0.84). The median (range) postoperative hospital stay was 10 (5-105) days for IMN and 10 (6-27) days for CON patients (P = 0.68). CONCLUSION: In patients undergoing LT, perioperative IMN did not provide significant benefits in terms of preoperative nutritional status or postoperative outcome.

The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid.

Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

Prenatal exposure to inflammatory conditions increases Cx43 and Panx1 unopposed channel opening and activation of astrocytes in the offspring effect on neuronal survival.

Several epidemiological studies indicate that children born from mothers exposed to infections during gestation, have an increased risk to develop neurological disorders, including schizophrenia, autism and cerebral palsy. Given that it is unknown if astrocytes and their crosstalk with neurons participate in the above mentioned brain pathologies, the aim of this work was to address if astroglial paracrine signaling mediated by Cx43 and Panx1 unopposed channels could be affected in the offspring of LPS-exposed dams during pregnancy. Ethidium uptake experiments showed that prenatal LPS-exposure increases the activity of astroglial Cx43 and Panx1 unopposed channels in the offspring. Induction of unopposed channel opening by prenatal LPS exposure depended on intracellular Ca2+ levels, cytokine production and activation of p38 MAP kinase/iNOS pathway. Biochemical assays and Fura-2AM/DAF-FM time-lapse fluorescence images revealed that astrocytes from the offspring of LPS-exposed dams displayed increased spontaneous Ca2+ dynamics and NO production, whereas iNOS levels and release of IL-1ß/TNF-α were also increased. Interestingly, we found that prenatal LPS exposure enhanced the release of ATP through astroglial Cx43 and Panx1 unopposed channels in the offspring, resulting in an increased neuronal death mediated by the activation of neuronal P2X7 receptors and Panx1 channels. Altogether, this evidence suggests that astroglial Cx43 and Panx1 unopposed channel opening induced by prenatal LPS exposure depended on the inflammatory activation profile and the activation pattern of astrocytes. The understanding of the mechanism underlying astrocyte-neuron crosstalk could contribute to the development of new strategies to ameliorate the brain abnormalities induced in the offspring by prenatal inflammation. GLIA 2015;63:2058-2072.

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins.

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.

Psychometric properties of the Cardiac Depression Scale: a systematic review.

BACKGROUND: The prevalence of depression is high in cardiac patients. Depression has a significant impact on quality of life, adherence to therapy, and an independent effect on prognosis. The Cardiac Depression Scale (CDS) is the only instrument designed to measure depression in cardiac patients. This study systematically reviewed the psychometric properties of the CDS for screening of depression in patients with coronary heart disease (CHD). METHODS: A search of MEDLINE, EMBASE, CINAHL Plus, PsycINFO, Scopus and Web of Science was performed using the search term Cardiac Depression Scale in the title or abstract. Eligible studies were those that assessed reliability, validity or diagnostic accuracy of the CDS in patients with CHD. Methodological quality was assessed using the QUADAS-2 and STARD. RESULTS: Most studies assessed the reliability and validity of the CDS: three studies assessed construct validity using factor analysis; six studies assessed the validity of the CDS with other measures of depression; and four studies assessed its diagnostic accuracy. However, some studies reported overlapping samples, which reduces confidence in their evaluation. CONCLUSION: This review finds the CDS to be a psychometrically sound measurement instrument for identifying mild, moderate and severe depression in cardiac populations.

The metabolic cofactor Coenzyme A enhances alternative macrophage activation via MyD88-linked signaling.

Metabolites and metabolic co-factors can shape the innate immune response, though the pathways by which these molecules adjust inflammation remain incompletely understood. Here we show that the metabolic cofactor Coenzyme A (CoA) enhances IL-4 driven alternative macrophage activation [m(IL-4)] in vitro and in vivo. Unexpectedly, we found that perturbations in intracellular CoA metabolism did not influence m(IL-4) differentiation. Rather, we discovered that exogenous CoA provides a weak TLR4 signal which primes macrophages for increased receptivity to IL-4 signals and resolution of inflammation via MyD88. Mechanistic studies revealed MyD88-linked signals prime for IL-4 responsiveness, in part, by reshaping chromatin accessibility to enhance transcription of IL-4-linked genes. The results identify CoA as a host metabolic co-factor that influences macrophage function through an extrinsic TLR4-dependent mechanism, and suggests that damage-associated molecular patterns (DAMPs) can prime macrophages for alternative activation and resolution of inflammation.

TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial.

In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.

What is the NCD service capacity and disruptions due to COVID-19? Results from the WHO non-communicable disease country capacity survey in the Americas region.

OBJECTIVE: This article presents the Americas regional results of the WHO non-communicable diseases (NCDs) Country Capacity Survey from 2019 to 2021, on NCD service capacity and disruptions from the COVID-19 pandemic. SETTING: Information on public sector primary care services for NCDs, and related technical inputs from 35 countries in the Americas region are provided. PARTICIPANTS: All Ministry of Health officials managing a national NCD programme, from a WHO Member State in the Americas region, were included throughout this study. Government health officials from countries that are not WHO Member States were excluded. OUTCOME MEASURES: The availability of evidence-based NCD guidelines, essential NCD medicines and basic technologies in primary care, cardiovascular disease risk stratification, cancer screening and palliative care services were measured in 2019, 2020 and 2021. NCD service interruptions, reassignments of NCD staff during the COVID-19 pandemic and mitigation strategies to reduce disruptions for NCD services were measured in 2020 and 2021. RESULTS: More than 50% of countries reported a lack of comprehensive package of NCD guidelines, essential medicines and related service inputs. Extensive disruptions in NCD services resulted from the pandemic, with only 12/35 countries (34%), reporting that outpatient NCD services were functioning normally. Ministry of Health staff were largely redirected to work on the COVID-19 response, either full time or partially, reducing the human resources available for NCD services. Six of 24 countries (25%) reported stock out of essential NCD medicines and/or diagnostics at health facilities which affected service continuity. Mitigation strategies to ensure continuity of care for people with NCDs were deployed in many countries and included triaging patients, telemedicine and teleconsultations, and electronic prescriptions and other novel prescribing practices. CONCLUSIONS: The findings from this regional survey suggest significant and sustained disruptions, affecting all countries regardless of the country's level of investments in healthcare or NCD burden.

Dendritic Cell Vaccination in Conjunction with a TLR Agonist Polarizes Interferon Immune Responses in Malignant Glioma Patients.

Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes after ATL-DC + TLR agonist administration. In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes. Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression. The results suggest that ATL-DC in conjunction with adjuvant poly-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01204684.

Modification of subcutaneous white adipose tissue inflammation by omega-3 fatty acids is limited in human obesity-a double blind, randomised clinical trial.

BACKGROUND: Obesity is associated with enhanced inflammation. However, investigation in human subcutaneous white adipose tissue (scWAT) is limited and the mechanisms by which inflammation occurs have not been well elucidated. Marine long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have anti-inflammatory actions and may reduce scWAT inflammation. METHODS: Subcutaneous white adipose tissue (scWAT) biopsies were collected from individuals living with obesity (n=45) and normal weight individuals (n=39) prior to and following a 12-week intervention with either 3 g/day of a fish oil concentrate (providing 1.1 g eicosapentaenoic acid (EPA) + 0.8 g docosahexaenoic acid (DHA)) or 3 g/day of corn oil. ScWAT fatty acid, oxylipin, and transcriptome profiles were assessed by gas chromatography, ultra-pure liquid chromatography tandem mass spectrometry, RNA sequencing and qRT-PCR, respectively. FINDINGS: Obesity was associated with greater scWAT inflammation demonstrated by lower concentrations of specialised pro-resolving mediators (SPMs) and hydroxy-DHA metabolites and an altered transcriptome with differential expression of genes involved in LC n-3 PUFA activation, oxylipin synthesis, inflammation, and immune response. Intervention with LC n-3 PUFAs increased their respective metabolites including the SPM precursor 14-hydroxy-DHA in normal weight individuals and decreased arachidonic acid derived metabolites and expression of genes involved in immune and inflammatory response with a greater effect in normal weight individuals. INTERPRETATION: Downregulated expression of genes responsible for fatty acid activation and metabolism may contribute to an inflammatory oxylipin profile and limit the effects of LC n-3 PUFAs in obesity. There may be a need for personalised LC n-3 PUFA supplementation based on obesity status. FUNDING: European Commission Seventh Framework Programme (Grant Number 244995) and Czech Academy of Sciences (Lumina quaeruntur LQ200111901).

Dysregulation of Subcutaneous White Adipose Tissue Inflammatory Environment Modelling in Non-Insulin Resistant Obesity and Responses to Omega-3 Fatty Acids - A Double Blind, Randomised Clinical Trial.

Background: Obesity is associated with enhanced lipid accumulation and the expansion of adipose tissue accompanied by hypoxia and inflammatory signalling. Investigation in human subcutaneous white adipose tissue (scWAT) in people living with obesity in which metabolic complications such as insulin resistance are yet to manifest is limited, and the mechanisms by which these processes are dysregulated are not well elucidated. Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have been shown to modulate the expression of genes associated with lipid accumulation and collagen deposition and reduce the number of inflammatory macrophages in adipose tissue from individuals with insulin resistance. Therefore, these lipids may have positive actions on obesity associated scWAT hypertrophy and inflammation. Methods: To evaluate obesity-associated tissue remodelling and responses to LC n-3 PUFAs, abdominal scWAT biopsies were collected from normal weight individuals and those living with obesity prior to and following 12-week intervention with marine LC n-3 PUFAs (1.1 g EPA + 0.8 g DHA daily). RNA sequencing, qRT-PCR, and histochemical staining were used to assess remodelling- and inflammatory-associated gene expression, tissue morphology and macrophage infiltration. Results: Obesity was associated with scWAT hypertrophy (P < 0.001), hypoxia, remodelling, and inflammatory macrophage infiltration (P = 0.023). Furthermore, we highlight the novel dysregulation of Wnt signalling in scWAT in non-insulin resistant obesity. LC n-3 PUFAs beneficially modulated the scWAT environment through downregulating the expression of genes associated with inflammatory and remodelling pathways (P <0.001), but there were altered outcomes in individuals living with obesity in comparison to normal weight individuals. Conclusion: Our data identify dysregulation of Wnt signalling, hypoxia, and hypertrophy, and enhanced macrophage infiltration in scWAT in non-insulin resistant obesity. LC n-3 PUFAs modulate some of these processes, especially in normal weight individuals which may be preventative and limit the development of restrictive and inflammatory scWAT in the development of obesity. We conclude that a higher dose or longer duration of LC n-3 PUFA intervention may be needed to reduce obesity-associated scWAT inflammation and promote tissue homeostasis. Clinical Trial Registration: www.isrctn.com, identifier ISRCTN96712688.

Histological characterization and quantification of newborn cells in the adult rodent brain.

This protocol describes the identification and characterization of newborn cells generated in the rodent brain after injury through birthdating with the thymidine analog 5-bromo-2'-deoxyuridine, followed by immunohistochemical labeling of fixed tissue sections. We also describe a software-assisted approach for automated detection and quantification of cells in large three-dimensional tissue volumes acquired using confocal microscopy. This approach facilitates the identification of low-frequency events that may be difficult to capture using manual counting methods, including stereology based on random sampling. For complete details on the use and execution of this protocol, please refer to Ermine et al. (2020).