Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription.Materials and methods:In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed.Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs.Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary

Nordiska slöjdpedagogers syn på hållbar utveckling som innehåll i slöjdundervisningen

Inom läroämnet slöjd har sparsamhet och återanvändning alltid varit i fokus, men i och med de globala utmaningarna om klimatfrågor har hållbar utveckling blivit aktuellt inom alla livsområden, utbildningsnivåer och läroämnen. Detta faktum ledde till att det nordiska nätverket SELAS (Sloyd Education, Learning And Sustainability) grundades år 2017. Syftet med nätverket är att bedriva grundforskning och utvecklingsarbete inom slöjdutbildning med hållbarhet som värde, kunskaper och färdigheter genom social, teknologisk, kulturell och ekonomisk hållbarhet. Pedagoger inom olika ämnen och utbildningsnivåer anses vara i nyckelposition att utbilda människor om hållbar utveckling och ge dem verktyg för att bidra till detta. I föreliggande artikel undersöks och diskuteras hållbar utveckling i relation till slöjdundervisning genom intervjuer med åtta nordiska slöjdpedagoger. Det övergripande syftet för studien är att utforska nordiska slöjdpedagogers, i huvudsak lärarutbildares, syn på hållbar utveckling i slöjdundervisningen. Avsikten är att erhålla kunskap om hur hållbar utveckling kan utgöra didaktiskt innehåll i slöjdundervisningen. Resultaten visar att hållbar utveckling kan på ett flertal olika sätt naturligt utgöra didaktiskt innehåll i slöjdundervisningen utgående från läroämnet slöjd, pedagogen i slöjd och slöjdaren. Sökord: didaktik, hållbar utveckling, slöjd, undervisning, NordenInom läroämnet slöjd har sparsamhet och återanvändning alltid varit i fokus, men i och med de globala utmaningarna om klimatfrågor har hållbar utveckling blivit aktuellt inom alla livsområden, utbildningsnivåer och läroämnen. Detta faktum ledde till att det nordiska nätverket SELAS (Sloyd Education, Learning And Sustainability) grundades år 2017. Syftet med nätverket är att bedriva grundforskning och utvecklingsarbete inom slöjdutbildning med hållbarhet som värde, kunskaper och färdigheter genom social, teknologisk, kulturell och ekonomisk hållbarhet. Pedagoger inom olika ämnen och utbildningsnivåer anses vara i nyckelposition att utbilda människor om hållbar utveckling och ge dem verktyg för att bidra till detta. I föreliggande artikel undersöks och diskuteras hållbar utveckling i relation till slöjdundervisning genom intervjuer med åtta nordiska slöjdpedagoger. Det övergripande syftet för studien är att utforska nordiska slöjdpedagogers, i huvudsak lärarutbildares, syn på hållbar utveckling i slöjdundervisningen. Avsikten är att erhålla kunskap om hur hållbar utveckling kan utgöra didaktiskt innehåll i slöjdundervisningen. Resultaten visar att hållbar utveckling kan på ett flertal olika sätt naturligt utgöra didaktiskt innehåll i slöjdundervisningen utgående från läroämnet slöjd, pedagogen i slöjd och slöjdaren. Sökord: didaktik, hållbar utveckling, slöjd, undervisning, Norde

Nordiska slöjdpedagogers syn på hållbar utveckling som innehåll i slöjdundervisningen

Inom läroämnet slöjd har sparsamhet och återanvändning alltid varit i fokus, men i och med de globala utmaningarna om klimatfrågor har hållbar utveckling blivit aktuellt inom alla livsområden, utbildningsnivåer och läroämnen. Detta faktum ledde till att det nordiska nätverket SELAS (Sloyd Education, Learning And Sustainability) grundades år 2017. Syftet med nätverket är att bedriva grundforskning och utvecklingsarbete inom slöjdutbildning med hållbarhet som värde, kunskaper och färdigheter genom social, teknologisk, kulturell och ekonomisk hållbarhet. Pedagoger inom olika ämnen och utbildningsnivåer anses vara i nyckelposition att utbilda människor om hållbar utveckling och ge dem verktyg för att bidra till detta. I föreliggande artikel undersöks och diskuteras hållbar utveckling i relation till slöjdundervisning genom intervjuer med åtta nordiska slöjdpedagoger. Det övergripande syftet för studien är att utforska nordiska slöjdpedagogers, i huvudsak lärarutbildares, syn på hållbar utveckling i slöjdundervisningen. Avsikten är att erhålla kunskap om hur hållbar utveckling kan utgöra didaktiskt innehåll i slöjdundervisningen. Resultaten visar att hållbar utveckling kan på ett flertal olika sätt naturligt utgöra didaktiskt innehåll i slöjdundervisningen utgående från läroämnet slöjd, pedagogen i slöjd och slöjdaren

DataSheet1_Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.docx

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription.Materials and methods:In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed.Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs.Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.</p

Glucosinolate Profiles and Phylogeny in Barbarea Compared to Other Tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), Based on a Library of Ion Trap HPLC-MS/MS Data of Reference Desulfoglucosinolates

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale

Fig. 3 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 3. HPLC-MS chromatograms of desulfoglucosinolates (dGSLs) prepared from glucosinolates (GSLs) in Planodes virginica (A) and Nasturtium officinale (B–C) seeds, showing qualitative similarities and quantitative contrasts. Major peaks (B) from N. officinale revealed many of the same GSLs as in A, but levels of EBAR (40R) were much lower while levels were much higher for the biosynthetic precursor PE (105). A focus on trace peaks from N. officinale (C) revealed sharp peaks representing a range of minor constituents. Due to the closely eluting peaks, the latter chromatograms (B–C) were made by combining extracted ion chromatograms corresponding to [M+Na]+ of the indicated dGSLs. In C, the m/z 366 signal of d105 was omitted to allow visualization of minor coeluting peaks. An asterisk after a peak number indicates tentative identification. HPLC-MS conditions as in Olsen et al. (2016). TIC, total ion chromatogram, EIC, extracted ion chromatogram.Published as part of &lt;i&gt;Agerbirk, Niels, Hansen, Cecilie Cetti, Olsen, Carl Erik, Kiefer, Christiane, Hauser, Thure P., Christensen, Stina, Jensen, Karen R., Ørgaard, Marian, Pattison, David I., Lange, Conny Bruun Asmussen, Cipollini, Don &amp; Koch, Marcus A., 2021, Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates, pp. 1-19 in Phytochemistry (112658) 185&lt;/i&gt; on page 8, DOI: 10.1016/j.phytochem.2021.112658, &lt;a href="http://zenodo.org/record/8259676"&gt;http://zenodo.org/record/8259676&lt;/a&gt

Glucosinolate profiles and phylogeny in <i>Barbarea</i> compared to other tribe Cardamineae (Brassicaceae) and <i>Reseda</i> (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale

Fig. 4 in Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates

Fig. 4. HPLC-MS chromatogram of desulfoglucosinolates (dGSLs) prepared from glucosinolates (GSLs) from leaves (A) and petioles (B) of horseradish (Armoracia rusticana), focusing on trace level GSLs. The chromatograms were made by combining extracted ion chromatograms corresponding to [M+Na]+ of the indicated dGSLs, from analyses that were much overloaded with respect to the dominating dGSL d107 from Pren. In panel A, an insert shows magnification of the chromatogram from 5.2 to 5.8 min. Neither suggested BAR nor EBAR were detectable. In panel B, only extracted ion chromatograms of m/z 382 (BAR/EBAR), 352 (BZ), 366 (PE), 380 (3PP), 394 (4PB), 408 (5PP at high tR and 6mSOh at 5.4 min), 422 (7mSOh) and 436 (8mSOo) are included. Unlabeled trace peaks did not exhibit a combination of tR and m/z suitable for any of the mentioned candidates. HPLC conditions as in Olsen et al. (2016). Panel A depicts analysis of the Copenhagen garden accession; panel B from the naturalized population at Lake Fures¨o. An asterisk after a peak number indicates tentative identification.Published as part of &lt;i&gt;Agerbirk, Niels, Hansen, Cecilie Cetti, Olsen, Carl Erik, Kiefer, Christiane, Hauser, Thure P., Christensen, Stina, Jensen, Karen R., Ørgaard, Marian, Pattison, David I., Lange, Conny Bruun Asmussen, Cipollini, Don &amp; Koch, Marcus A., 2021, Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates, pp. 1-19 in Phytochemistry (112658) 185&lt;/i&gt; on page 10, DOI: 10.1016/j.phytochem.2021.112658, &lt;a href="http://zenodo.org/record/8259676"&gt;http://zenodo.org/record/8259676&lt;/a&gt

One thousand plant transcriptomes and the phylogenomics of green plants

Abstract: Green plants (Viridiplantae) include around 450,000–500,000 species1, 2 of great diversity and have important roles in terrestrial and aquatic ecosystems. Here, as part of the One Thousand Plant Transcriptomes Initiative, we sequenced the vegetative transcriptomes of 1,124 species that span the diversity of plants in a broad sense (Archaeplastida), including green plants (Viridiplantae), glaucophytes (Glaucophyta) and red algae (Rhodophyta). Our analysis provides a robust phylogenomic framework for examining the evolution of green plants. Most inferred species relationships are well supported across multiple species tree and supermatrix analyses, but discordance among plastid and nuclear gene trees at a few important nodes highlights the complexity of plant genome evolution, including polyploidy, periods of rapid speciation, and extinction. Incomplete sorting of ancestral variation, polyploidization and massive expansions of gene families punctuate the evolutionary history of green plants. Notably, we find that large expansions of gene families preceded the origins of green plants, land plants and vascular plants, whereas whole-genome duplications are inferred to have occurred repeatedly throughout the evolution of flowering plants and ferns. The increasing availability of high-quality plant genome sequences and advances in functional genomics are enabling research on genome evolution across the green tree of life