The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ...ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.
Display omitted
► Ribonucleotides are the most common nucleotide base lesion in the mouse genome ► RNase H2 is a key genome surveillance enzyme required for removal of nucleotides ► RNase H2 is essential for mammalian development ► Without RNase H2, cells exhibit genome instability and p53 pathway activation
DNA polymerases can incorporate more than a million ribonucleotides into replicating mouse DNA in each cell, making ribonucleotides the most abundant kind of DNA lesion. RNase H2, which removes this “damage,” has an essential role in genome surveillance and is required for embryonic development.
Archaea represent a significant fraction of Earth's biodiversity, yet they remain much less well understood than Bacteria. Gene surveys, a few metagenomic studies, and some single-cell sequencing ...projects have revealed numerous little-studied archaeal phyla. Certain lineages appear to branch deeply and may be part of a major phylum radiation. The structure of this radiation and the physiology of the organisms remain almost unknown.
We used genome-resolved metagenomic analyses to investigate the diversity, genomes sizes, metabolic capacities, and potential roles of Archaea in terrestrial subsurface biogeochemical cycles. We sequenced DNA from complex sediment and planktonic consortia from an aquifer adjacent to the Colorado River (USA) and reconstructed the first complete genomes for Archaea using cultivation-independent methods. To provide taxonomic context, we analyzed an additional 151 newly sampled archaeal sequences. We resolved two new phyla within a major, apparently deep-branching group of phyla (a superphylum). The organisms have small genomes, and metabolic predictions indicate that their primary contributions to Earth's biogeochemical cycles involve carbon and hydrogen metabolism, probably associated with symbiotic and/or fermentation-based lifestyles.
The results dramatically expand genomic sampling of the domain Archaea and clarify taxonomic designations within a major superphylum. This study, in combination with recently published work on bacterial phyla lacking cultivated representatives, reveals a fascinating phenomenon of major radiations of organisms with small genomes, novel proteome composition, and strong interdependence in both domains.
•HPLC-DAD cannabinoids quantitation in large variety of commercial cannabis products.•Foods, candies, beverages, topicals, vapes/eliquids, oral supplements.•11 cannabis cannabinoids resolved with ...mixed C18-aromatic stationary phase.•Extensive method validation for CBD, Δ9-THC, CBDA, THCA, and CBN.
Quantitative analysis for the cannabis cannabinoids such as cannabidiol and Δ9-tetrahydrocannabinol in commercial products is necessary for evaluating label information, and assessing dosages and exposures when the products are consumed. Herein is presented a broadly applicable HPLC-DAD method for the determination of cannabis cannabinoids in commercial consumer products and traditional plant-related substances. The current method provides chromatographic resolution of 11 cannabinoids using a commercial, mixed C18-aromatic functionality stationary phase. The method uses 95% or pure ethanol for extraction, and certain modifications which address specific matrix types are detailed herein. Extensive method validation including precision and accuracy was conducted for five cannabinoids of primary interest (CBD, Δ9-THC, CBDA, THCA, and CBN). UV detection provided excellent sensitivity with limits of quantitation (LOQs) of 10μg/g across cannabinoids. The method was applied to about 60 commercial products representing diverse product types and a broad range of cannabinoids amounts (0.01–350mg/g).
Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid ...tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.
•Cancer cells of many tissues express an abnormal glycoform of MUC1, Tn-MUC1•Normal human tissue does not express detectable Tn-MUC1 on the cellular surface•CAR T cells are engineered to target Tn-MUC1 lyse tumor cells in vitro and in vivo•Abnormal glycoform epitopes are valid clinical targets for CAR T cells
Posey and colleagues developed a CAR T cell therapy to break immune tolerance to solid tumors by targeting an aberrantly glycosylated, cancer-specific glycoprotein in multiple cancer histotypes and demonstrated efficacy and safety in tumors as diverse as leukemia and pancreatic cancer.
Sickle cell disease (SCD) disproportionately affects Black or African American persons in the United States and can cause multisystem organ damage and reduced lifespan. Among 178 persons with SCD in ...the United States who were reported to an SCD-coronavirus disease case registry, 122 (69%) were hospitalized and 13 (7%) died.
Although there is much research detailing the pedagogical constraints of high-stakes testing (HST), there is less that examines teachers’ practices within and beyond its control. This multiple case ...study analyzes ethnographic data to explore two teachers’ practices in an urban context where HST was relevant. Drawing on Foucault’s conceptualization of a plague-stricken town, we explore the mobilization of disciplinary power associated with HST. We then examine teachers’ agency as they taught beyond the administrative gaze. We found that teachers sometimes complied with administrative mandates while also articulating tensions of providing access and actively resisting/critiquing the test.
•Cannabinoids GC–MS identification in large variety of commercial cannabis products.•Foods, candies, beverages, topicals, vapes/eliquids, oral supplements.•11 cannabis cannabinoids resolved with 35% ...silphenylene stationary phase.•Common interferents minimized using selective extraction and sample pretreatment.•Substantial method validation for CBD, Δ9-THC, CBDA, THCA, and CBN.
The recent surge in the sale of cannabis-based consumer products in the US includes foods, candies, beverages, topicals, vapes/eliquids, oral supplements in various forms, recreational marijuana plants, and plant extracts or preparations. The wide variety of product and sample types has resulted in a host of new matrix interferences when conducting qualitative testing for the cannabis cannabinoids such as cannabidiol and d9-tetrahydrocannabinol. A qualitative GC–MS method is presented in this work, which uses a commercial 35% silphenylene phase to provide chromatographic resolution for 11 target cannabinoids as their trimethylsilyl derivatives (CBD, CBDA, d9THC, THCA, CBN, d8THC, CBG, CBGA, CBDV, THCV, and CBC). The method uses variants of ethanol- and acetonitrile-based extractants to successfully minimize or eliminate several types of interferents, and also provides protocols to address specific interferents such as glycerin and lactose. Method validation included spike/recovery for five cannabinoids of primary interest (spiking level 50μg/g) from a series of edible oils, foods, beverages, candies, topicals, oral OTC pharmaceuticals, glycerin, and propylene glycol. The minimum detectable concentration was established as 1.0μg/g. The method was applied to about sixty diverse commercial products, as well as to recreational marijuana plants, plant preparations, hempseed oils, and dronabinol capsules.
•Neoliberalism constructs ideological and material barriers to classroom silence•Student silence is perceived negatively within a neoliberal framework•Student silence is constrained by standardizing ...neoliberal policies and practices•Silence can be used by teachers as a humanizing pedagogical tool
Attending to both the political and pedagogical dimensions of silence in schools, this article engages in a close analysis of the silences constructed within an interaction between one first-grade student and his teacher in order to build theory regarding how silence is constructed in the classroom. Drawing on theories of neoliberalism as well as humanizing pedagogy, it presents multiple readings of this interaction to illustrate how interpretation of silence is shaped by ideology. Further, it emphasizes the mediating role of mandated standardized curriculum in the production of silence in this interaction. Through this analysis, it argues that neoliberalism creates both ideological and material barriers to the practice of silence in contemporary classrooms, while also suggesting how silence might be strategically used by teachers as a humanizing pedagogical resource.
VX-445 is a next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector designed to restore Phe508del CFTR protein function in patients with cystic fibrosis when administered ...with tezacaftor and ivacaftor (VX-445-tezacaftor-ivacaftor).
We evaluated the effects of VX-445-tezacaftor-ivacaftor on Phe508del CFTR protein processing, trafficking, and chloride transport in human bronchial epithelial cells. On the basis of in vitro activity, a randomized, placebo-controlled, double-blind, dose-ranging, phase 2 trial was conducted to evaluate oral VX-445-tezacaftor-ivacaftor in patients heterozygous for the Phe508del CFTR mutation and a minimal-function mutation (Phe508del-MF) and in patients homozygous for the Phe508del CFTR mutation (Phe508del-Phe508del) after tezacaftor-ivacaftor run-in. Primary end points were safety and absolute change in percentage of predicted forced expiratory volume in 1 second (FEV
) from baseline.
In vitro, VX-445-tezacaftor-ivacaftor significantly improved Phe508del CFTR protein processing, trafficking, and chloride transport to a greater extent than any two of these agents in dual combination. In patients with cystic fibrosis, VX-445-tezacaftor-ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. The treatment also resulted in an increased percentage of predicted FEV
of up to 13.8 points in the Phe508del-MF group (P<0.001). In patients in the Phe508del-Phe508del group, who were already receiving tezacaftor-ivacaftor, the addition of VX-445 resulted in an 11.0-point increase in the percentage of predicted FEV
(P<0.001). In both groups, there was a decrease in sweat chloride concentrations and improvement in the respiratory domain score on the Cystic Fibrosis Questionnaire-Revised.
The use of VX-445-tezacaftor-ivacaftor to target Phe508del CFTR protein resulted in increased CFTR function in vitro and translated to improvements in patients with cystic fibrosis with one or two Phe508del alleles. This approach has the potential to treat the underlying cause of cystic fibrosis in approximately 90% of patients. (Funded by Vertex Pharmaceuticals; VX16-445-001 ClinicalTrials.gov number, NCT03227471 ; and EudraCT number, 2017-000797-11 .).
In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require ...an adaptive immune system
. For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing
, prevent developmental malformations
and replace old tissues during regeneration
. At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins
. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated
. However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed
. Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease
. However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage
. This would be undesirable for humans because it might make tumours more aggressive
. It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.
PDF
