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Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm 5 ), 5-methoxycarbonylmethyl (mcm 5 ), or 5-methoxycarbonylhydroxymethyl (mchm 5 ) side-chain. The presence of these side-chains allows proper pairing with cognate codons, and they are particularly important in tRNA species where the U 34 residue is also modified with a 2-thio (s 2 ) group. The first step in the synthesis of the ncm 5 , mcm 5 , and mchm 5 side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1 , which encodes the cytosolic Cu, Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-modified U 34 residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNA Phe Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells., (© 2024 Xu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Modifications at the wobble position (position 34) of tRNA facilitate interactions that enable or stabilize non-Watson-Crick base pairs. In bacterial tRNA, 5-hydroxyuridine (ho 5 U) derivatives xo 5 U [x: methyl (mo 5 U), carboxymethyl (cmo 5 U), and methoxycarbonylmethyl (mcmo 5 U)] present at the wobble positions of tRNAs are responsible for the recognition of NYN codon families. These modifications of U34 allow base-pairing not only with A and G but also with U, and in some cases, C. mo 5 U was originally found in Gram-positive bacteria, and cmo 5 U and mcmo 5 U were found in Gram-negative bacteria. tRNAs of Mycoplasma species, mitochondria, and chloroplasts adopt four-way decoding in which unmodified U34 recognizes codons ending in A, G, C, and U. Lactobacillus casei , Gram-positive bacteria, and lactic acid bacteria lack the modification enzyme genes for xo 5 U biosynthesis. Nevertheless, L. casei has only one type of tRNA Val with the anticodon UAC [tRNA Val (UAC)]. However, the genome of L. casei encodes an undetermined tRNA (tRNA Und ) gene, and the sequence corresponding to the anticodon region is GAC. Here, we confirm that U34 in L. casei tRNA Val is unmodified and that there is no tRNA Und expression in the cells. In addition, in vitro transcribed tRNA Und was not aminoacylated by L. casei valyl-tRNA synthetase, suggesting that tRNA Und is not able to accept valine, even if expressed in cells. Correspondingly, native tRNA Val (UAC) with unmodified U34 bound to all four valine codons in the ribosome A site. This suggests that L. casei tRNA Val decodes all valine codons by four-way decoding, similarly to tRNAs from Mycoplasma species, mitochondria, and chloroplasts., (© 2024 Sugita et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA for several decades. Here, we demonstrate that 5hmU contents vary among different dinoflagellates and are generated through thymidine hydroxylation. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homolog, catalyzing 5hmU production using both in vivo and in vitro biochemical assays. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae , we depicted a comprehensive 5hmU landscape and found that 5hmU loci are significantly enriched in repeat elements. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements, implying that 5hmU appears to serve as an epigenetic mark for silencing transposon. Together, our results revealed the biogenesis, genome-wide landscape, and molecular function of dinoflagellate 5hmU, providing mechanistic insight into the function of this enigmatic DNA mark., Competing Interests: Competing interests statement:The authors declare no competing interest.

Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA-modifying enzyme that methylates wobble uridines in a subset of tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila , we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit reduced protein synthesis in the nervous system, including a specific decrease in selenoprotein levels. Either loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 null animals also exhibit associative memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA wobble uridine modification in redox homeostasis in the developing nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability., Competing Interests: Competing interests statement:The authors declare no competing interest.

Echinochloa phyllopogon is a noxious weed that can harm rice over prolonged periods. Recently, a penoxsulam-resistant variant of E. phyllopogon with a mutation in the acetolactate synthase (ALS) gene was collected in Northeastern China. In the present study, the molecular mechanism underlying herbicide resistance in mutant populations was evaluated. The GR 50 and IC 50 values of the herbicide-resistant mutant 1-11 were 27.0- and 21.4-fold higher than those of the susceptible population 2-31, respectively. In addition, pre-application of malathion reduced the GR 50 value of the resistant population. Additionally, mutant populations developed cross-resistance to other ALS inhibitors. E. phyllopogon ALS sequencing showed a Trp-574-Leu mutation in ALS2 variant 1-11. Molecular docking showed that the Trp-574-Leu substitution reduced the number of hydrogen bonds and altered the interaction between penoxsulam and ALS2. Transgenic Arabidopsis plants harboring the ALS2 mutant gene also showed resistance to penoxsulam and other ALS inhibitors. Overall, our study demonstrated that the Trp-574-Leu mutation and P450-mediated metabolic resistance lead to the cross-resistance of E. phyllopogon to ALS inhibitors., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

This study compared decitabine exposure when administered IV (DEC-IV) at a dose of 20 mg/m 2 for 5-days with orally administered decitabine with cedazuridine (DEC-C), as well as the clinical efficacy and safety of DEC-C in patients with acute myeloid leukaemia (AML) who were ineligible for intensive induction chemotherapy. In all, 89 patients were randomised 1:1 to DEC-IV or oral DEC-C (days 1-5 in a 28-day treatment cycle), followed by 5 days of the other formulation in the next treatment cycle. All patients received oral DEC-C for subsequent treatment cycles until treatment discontinuation. Equivalent systemic decitabine exposures were demonstrated (5-day area under the curve ratio between the two decitabine formulations of 99.64 [90% confidence interval 91.23%, 108.80%]). Demethylation rates also were similar (≤1.1% difference). Median overall survival (OS), clinical response and safety profile with oral DEC-C were consistent with those previously observed with DEC-IV. Next-generation sequencing was performed to identify molecular abnormalities that impact OS and TP53 mutations were associated with a poor outcome. These findings support the use of oral DEC-C in patients with AML., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

In a randomized crossover study involving 89 patients with acute myeloid leukaemia ineligible for intensive chemotherapy, Geissler et al. compared intravenous decitabine and oral decitabine-cedazuridine. The pharmacokinetics and pharmacodynamics of the two formulations were similar. The clinical efficacy of oral decitabine-cedazuridine was consistent with historical data of intravenous decitabine. Commentary on: Geissler et al. Oral decitabine/cedazuridine versus intravenous decitabine for acute myeloid leukaemia: A randomised, crossover, registration, pharmacokinetics study. Br J Haematol 2024; 205:1734-1745., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Chemotherapies are standard care for most cancer types. Pyrimidine analogs including 5-fluorouracil, cytosine arabinoside, 5-azacytidine, and gemcitabine are effective drugs that are utilized as part of a number of anticancer regimens. However, their lack of cell-specificity results in severe side effects. Therefore, there is a capacity to improve the efficacy of such therapies, while decreasing unwanted side effects. Here, we report that while 5-fluorocytosine is not chemotherapeutic in itself, incorporated into a ribonucleoside and more importantly into an RNA oligonucleotide, it induces cytotoxic effects on cancer cells in vitro . Interestingly, these effects are rescued by both uridine and thymidine. Similarly, in-vitro 2'-deoxy-5-fluorocytidine inhibits the growth of tumor cells but has the advantage of being less toxic to human primary cells compared with 5-fluorocytidine, suggesting that the deoxyribonucleoside could exhibit less side-effects in vivo . Thus, this work indicates that the potency of 5-fluorocytidine and 2'-deoxy-5-fluorocytidine should be further explored. In particular, oligonucleotides incorporating 5-fluorocytosine could be novel chemotherapeutic drugs that could be formulated in cancer-specific particles for safe and efficacious cancer treatments., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Molnupiravir (MO) is a pyrimidine nucleoside anti-SARS-CoV-2 drug. MO treatment could cause mild liver injury. However, the underlying mechanism of MO-induced liver injury and the metabolic pathway of MO in vivo are unclear. In this study, metabolomics analysis and molecular biology methods were used to explore these issues. Through metabolomics analysis, it was found that the homeostasis of pyrimidine, purine, lysophosphatidylcholine (LPC), and amino acids in mice was destroyed after MO treatment. A total of 80 changed metabolites were detected. Among these changed metabolites, 4-ethylphenyl sulfate, dihydrouracil, and LPC 20:0 was related to the elevation of alkaline phosphatase (ALP), interleukin-6 (IL6), and nuclear factor kappa-B (NF-κB). The levels of 4-ethylphenyl sulfate, dihydrouracil, and LPC 20:0 in plasma were positively correlated with their levels in the liver, suggesting that these metabolites were associated with MO-induced liver injury. MO treatment could increase NHC and cytidine levels, activate cytidine deaminase (CDA), and increase LPC levels. CDA and LPC could increase the mRNA expression level of toll-like receptor (TLR). The current study indicated that the elevation of hepatic TLR may be an important reason for MO leading to the liver injury., (© 2024 John Wiley & Sons Ltd.)

RNA 5-methyluridine (m5U) sites play a significant role in understanding RNA modifications, which influence numerous biological processes such as gene expression and cellular functioning. Consequently, the identification of m5U sites can play a vital role in the integrity, structure, and function of RNA molecules. Therefore, this study introduces GRUpred-m5U, a novel deep learning-based framework based on a gated recurrent unit in mature RNA and full transcript RNA datasets. We used three descriptor groups: nucleic acid composition, pseudo nucleic acid composition, and physicochemical properties, which include five feature extraction methods ENAC, Kmer, DPCP, DPCP type 2, and PseDNC. Initially, we aggregated all the feature extraction methods and created a new merged set. Three hybrid models were developed employing deep-learning methods and evaluated through 10-fold cross-validation with seven evaluation metrics. After a comprehensive evaluation, the GRUpred-m5U model outperformed the other applied models, obtaining 98.41% and 96.70% accuracy on the two datasets, respectively. To our knowledge, the proposed model outperformed all the existing state-of-the-art technology. The proposed supervised machine learning model was evaluated using unsupervised machine learning techniques such as principal component analysis (PCA), and it was observed that the proposed method provided a valid performance for identifying m5U. Considering its multi-layered construction, the GRUpred-m5U model has tremendous potential for future applications in the biological industry. The model, which consisted of neurons processing complicated input, excelled at pattern recognition and produced reliable results. Despite its greater size, the model obtained accurate results, essential in detecting m5U., (© 2024. The Author(s).)

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits-the potent influence of Ψ55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. Growing cells under nutrient depleted conditions also revealed a novel condition-specific increase in m1A58 modification on some tRNAs. A global and isoacceptor-specific classification strategy was developed to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Acute hepatic porphyria is a group of multisystem disorders of which acute intermittent porphyria is the most common subtype. Givosiran, a subcutaneously administered RNA interference therapeutic targeting liver ALAS mRNA, is approved for treating these disorders. This Phase 1/2 open-label extension study (NCT02949830) evaluated the long-term safety and efficacy of givosiran in adults with acute intermittent porphyria, with follow-up of up to 48 months, which is the longest follow-up of givosiran treatment to date. Participants were adults aged 18-65 years who completed part C of the Phase 1 givosiran study (NCT2452372)., Methods: Enrollees received givosiran for up to 48 months. Primary and secondary endpoints included the incidence of adverse events, changes in urinary delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) levels, annualized rate of porphyria attacks, and annualized hemin use. Quality of life was assessed using the EQ-5D-5L instrument as an exploratory endpoint., Results: Sixteen patients (median age: 39.5 years) participated. Common adverse events included abdominal pain, nasopharyngitis, and nausea (50% each), with injection-site erythema (38%) and injection-site pruritus (25%) noted as frequent treatment-related reactions. Givosiran therapy reduced annualized rates of porphyria attacks and hemin use by 97% and 96%, respectively. From months > 33 to 48, all patients were free from attacks requiring significant medical intervention and did not use hemin. There were substantial reductions in median urinary ALA and PBG of 95% and 98%, respectively. Additionally, a clinically meaningful improvement in quality of life was observed., Conclusions: In the longest follow-up of givosiran-treated patients reported to date, the therapy maintained an acceptable safety profile and demonstrated sustained improvements in clinical outcomes over 4 years in patients with acute intermittent porphyria., (© 2024. The Author(s).)

Human cytoplasmic tRNAs contain dihydrouridine modifications at positions 16 and 17 (D16/D17). The enzyme responsible for D16/D17 formation and its cellular roles remain elusive. Here, we identify DUS1L as the human tRNA D16/D17 writer. DUS1L knockout in the glioblastoma cell lines LNZ308 and U87 causes loss of D16/D17. D formation is reconstituted in vitro using recombinant DUS1L in the presence of NADPH or NADH. DUS1L knockout/overexpression in LNZ308 cells shows that DUS1L supports cell growth. Moreover, higher DUS1L expression in glioma patients is associated with poorer prognosis. Upon vector-mediated DUS1L overexpression in LNZ308 cells, 5' and 3' processing of precursor tRNA Tyr(GUA) is inhibited, resulting in a reduced mature tRNA Tyr(GUA) level, reduced translation of the tyrosine codons UAC and UAU, and reduced translational readthrough of the near-cognate stop codons UAA and UAG. Moreover, DUS1L overexpression increases the amounts of several D16/D17-containing tRNAs and total cellular translation. Our study identifies a human dihydrouridine writer, providing the foundation to study its roles in health and disease., (© 2024. The Author(s).)

In this article, we describe the synthesis of N 3 -methyluridine (m 3 U) and 2'-O-alkyl/2'-fluoro-N 3 -methyluridine (2'-O-alkyl/2'-F-m 3 U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2'-O-alkyl-m 3 U phosphoramidite starts with commercially available uridine to achieve a tritylated m 3 U intermediate, followed by 2'-O-alkylation and finally phosphitylation. Synthesis of the 2'-F-m 3 U phosphoramidite is obtained from a commercially available 2'-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2'-position of m 3 U that can be employed in numerous nucleic acid-based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2'-O-alkyl-N 3 -methyluridine analogs and their corresponding phosphoramidites Alternate Protocol 1: Synthesis of 2'-O-TBDMS-N 3 -methyluridine and its phosphoramidite Alternate Protocol 2: Synthesis of 2'-fluoro-N 3 -methyluridine and its phosphoramidite Basic Protocol 2: Solid-phase synthesis of N 3 -methyluridine-modified DNA and RNA oligonucleotides., (© 2024 Wiley Periodicals LLC.)

We report the discovery and characterization of antioxidative effects of uridine linked with three dipeptide motifs against DNA oxidation induced by peroxyl radicals. First, the dipeptide motifs are constructed by using the Ugi four-component reaction (Ugi 4CR), in which caffeic, ferulic, sinapic, and syringic acids are used as the carboxylic acid resources, vanillin, benzaldehyde, and p -hydroxybenzaldehyde are used as the aldehyde resources, tyramine- and dopamine-related isocyanides as well as ethyl isocyanoacetate are used as the isocyanide resources, and 2-( p -aminophenyl)ethanol is used as the amine component. We found that the antioxidative effects of the Ugi 4CR products are 1.3-2.8 times higher than those of caffeic, ferulic, sinapic, and syringic acids in the protection of DNA against peroxyl radical-induced oxidation. Moreover, when three Ugi 4CR products are linked with three hydroxyl groups of uridine by using three succinic anhydrides as the linkage, the inhibitory effects of the afforded uridine-dipeptide hybrids against the DNA oxidation increase 4.4-8.9 times (>3 times) compared to that of the Ugi 4CR product. This is due to the hybrid structure consisting of uridine and three motifs of the Ugi 4CR product enabling binding with the DNA strand more efficiently and quenching free radicals more rapidly. Therefore, the hybrid structure constructed by the nucleoside with antioxidative dipeptides offers an additional advantage for protecting DNA against radical-induced oxidation.

RNA editing pathway is a validated target in kinetoplastid parasites ( Trypanosoma brucei , Trypanosoma cruzi , and Leishmania spp.) that cause severe diseases in humans and livestock. An essential large protein complex, the editosome, mediates uridine insertion and deletion in RNA editing through a stepwise process. This study details the discovery of editosome inhibitors by screening a library of widely used human drugs using our previously developed in vitro biochemical Ribozyme Insertion Deletion Editing (RIDE) assay. Subsequent studies on the mode of action of the identified hits and hit expansion efforts unveiled compounds that interfere with RNA-editosome interactions and novel ligase inhibitors with IC 50 values in the low micromolar range. Docking studies on the ligase demonstrated similar binding characteristics for ATP and our novel epigallocatechin gallate inhibitor. The inhibitors demonstrated potent trypanocidal activity and are promising candidates for drug repurposing due to their lack of cytotoxic effects. Further studies are necessary to validate these targets using more definitive gene-editing techniques and to enhance the safety profile.

Background: Outcomes are dismal for patients with myelofibrosis (MF) who are no longer responsive to JAK2 inhibitors (JAKi) and/or have increasing blast cell numbers. Although prior reports have suggested the benefits of intravenous decitabine (DAC) combined with ruxolitinib for patients with Myeloproliferative Neoplasm (MPN) accelerated/blast phase (AP/BP), decitabine-cedazuridine (DEC-C), an oral fixed-dose combination providing equivalent pharmacokinetic exposure, has not been evaluated in MF., Methods: We conducted a retrospective analysis of 14 patients with high-risk MF refractory to ruxolitinib or MPN-AP (10-19% blasts) treated with DEC-C +/- JAKi at Mount Sinai Hospital from 2021 to 2024., Results: The cohort was elderly (median age,76 years) and almost uniformly possessed high risk mutations with 13 of the 14 patients progressing on JAKi therapy. With a median follow-up of 9.4 months, the median overall survival (OS) was 29 months for the entire cohort. Median OS was 10.8 months for MPN-AP and was not reached for ruxolitinib refractory MF patients. All patients (n = 9) receiving > 4 cycles of DEC-C had clinical benefit exemplified by a reduction in blast cell numbers, spleen size, and lack of progression to MPN-BP (78%). Furthermore, 3/14 patients proceeded to allogeneic stem cell transplant. Myelosuppression was a common adverse event which was managed by reducing the number of days of administration of DEC-C from 5 to 3 per cycle., Conclusions: This report demonstrates the feasibility, tolerability, and clinical benefit of an exclusively ambulatory regimen for high-risk, elderly patients with advanced MF which warrants further evaluation in a prospective clinical trial., Competing Interests: Disclosures D.T. receives contracted research funding paid to his institution from Sobi, Astellas Pharma, Gilead and Cogent Biosciences and consulting fees from Novartis, AbbVie, Sierra Oncology, GSK and Cogent Biosciences. J.M. receives research funding paid to his institution from Incyte, Novartis, BMS, CTI/Sobi, Abbvie, Geron, Kartos and PharmaEssentia. Consulting fees paid to me by Incyte, Novartis, CTI/Sobi, Abbvie, Karyopharm, Kartos, Geron, GSK, Sumitomo, BMS, MorphoSys, Galecto, Pfizer, Merck, Roche and PharmaEssentia. R.H. received research grant funding through his institution from the following sources: Citi BioPharma Corp, Curis, Inc., Dexcel Pharma Technologies Ltd. Genentech, Kartos Therapeutics, Karyopharm Therapeutics, Inc., Kymera Therapeutics, OncoMyx Therapeutics, Novartis, Protagonist Therapeutics, Repare Therapeutics, Translational Drug Development (TD2), Turning Point Therapeutics, and the NIH/NCI. Additionally, he has received funding as an advisor/consultant from the following sources: AbbVie, Ionis Pharmaceuticals, Protagonist Therapeutics, Silence Therapeutics, GlycoMimetics, Dexcel Pharma Technologies LTD, Sumitomo Pharma Oncology, and Cellenkos, Inc. Finally, he holds a share of a patent filed by Dompé farmaceutici S.p.A. Y.Z.G. received research grant funding through her institution from the following sources: Protagonist Therapeutics, Repare Therapeutics, and the NIH/NIDDK. YZG has received funding as an advisor/consultant from the following sources: Bay Clinical, Takeda, Denali, Ionis Pharmaceuticals, Protagonist Therapeutics. No other relevant financial disclosures for other authors., (Copyright © 2024. Published by Elsevier Inc.)

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Eukaryotic RNA synthesis and degradation are intricately regulated, impacting on gene expression dynamics. RNA stability varies in individual transcripts and is modulated by trans-acting factors such as microRNAs, long noncoding RNAs, and RNA-binding proteins, which determine protein output and subsequent cellular processes. To measure RNA decay rate, accurate and reliable methodologies are essential in the field of RNA biology. Transcription inhibition and metabolic labeling enable comprehensive investigations on RNA decay, offering critical insights into dynamic regulation of RNA decay. Transcription shut-off has been employed widely by using various approaches, such as treatment with chemical inhibitors or generation of temperature-sensitive mutants of RNA polymerases. However, it has limitations, providing a static view and lacking real-time dynamics as well as precise measurement of decay rate. Metabolic labeling, a method of incorporating modified nucleotides into RNA transcripts, complements shut-off approaches, allowing selective monitoring of newly synthesized RNA and tracing decay intermediates. The purpose of the protocol described in this chapter is to assess the kinetics and statics of newly synthesized RNA and its decay by 5-ethynyl uridine labeling., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The gut microbiome can metabolise hundreds of drugs, potentially affecting their bioavailability and pharmacological effect. As most gut bacteria reside in the colon, drugs that reach the colon in significant proportions may be most impacted by microbiome metabolism. In this study the anti-colorectal cancer drug trifluridine was used as a model drug for characterising metabolism by the colonic microbiota, identifying correlations between bacterial species and individuals' rates of microbiome drug inactivation, and developing strategies to prevent drug inactivation following targeted colonic delivery. High performance liquid chromatography and ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry demonstrated trifluridine's variable and multi-route metabolism by the faecal microbiota sourced from six healthy humans. Here, four drug metabolites were linked to the microbiome for the first time. Metagenomic sequencing of the human microbiota samples revealed their composition, which facilitated prediction of individual donors' microbial trifluridine inactivation. Notably, the abundance of Clostridium perfringens strongly correlated with the extent of trifluridine inactivation by microbiota samples after 2 hours (R 2 = 0.8966). Finally, several strategies were trialled for the prevention of microbial trifluridine metabolism. It was shown that uridine, a safe and well-tolerated molecule, significantly reduced the microbiota's metabolism of trifluridine by acting as a competitive enzyme inhibitor. Further, uridine was found to provide prebiotic effects. The findings in this study greatly expand knowledge on trifluridine's interactions with the gut microbiome and provide valuable insights for investigating the microbiome metabolism of other drugs. The results demonstrate how protection strategies could enhance the colonic stability of microbiome-sensitive drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work was conducted before Laura E. McCoubrey joined GSK and as such this work does not reflect the views of GSK., (Copyright © 2024. Published by Elsevier B.V.)

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives., Competing Interests: Competing interests statement:The authors declare no competing interest.

We recently reported the properties of RNA hairpins constrained by a dimethylene (DME) disulfide (S-S) linker incorporated between two adjacent nucleosides in the loop and showed that this linker locked the hairpin conformation thus disturbing the duplex/hairpin equilibrium. We have now investigated the influence of the length of the linker and synthesized oligoribonucleotides containing diethylene (DEE) and dipropylene (DPE) S-S bridges. This was achieved via the preparation of building blocks, namely 2'-O-acetylthioethyl (2'-O-AcSE) and 2'-O-acetylthiopropyl (2'-O-AcSP) uridine phosphoramidites, which were successfully incorporated into RNA sequences. Thermal denaturation analysis revealed that the DEE and DPE disulfide bridges destabilize RNA duplexes but do not disrupt the hairpin conformation. Furthermore, our investigation of the duplex/hairpin equilibrium indicated that sequences modified with DME and DEE S-S linkers predominantly lock the hairpin form, whereas the DPE S-S linker provides flexibility. These findings highlight the potential of S-S linkers to study RNA interactions., (© 2023 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In this report, we show that a very common modification (especially in tRNA), dihydrouridine, was efficiently produced by photoreduction of the canonical pyrimidine ribonucleoside, uridine in formamide. Formamide not only acts as a solvent in this reaction, but also as the reductant. The other three components of the canonical alphabet (C, A, G) remained intact under the same conditions, suggesting that dihydrouridine might have coexisted with all four canonical RNA nucleosides (C, U, A, G) at the dawn of life.

Dengue is an important arboviral infection worldwide for which presently there is no specific medicine. Evidence suggests there are four serotypes of dengue virus (DENV1-4), of which DENV 2 is considered to cause the most sever dengue. Therefore, this study was aimed to develop the new uridine derivatives (NUDs) against dengue virus (DENV 2). In current study 2-(3,4-dihydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-4-((substituted cyclohexa-2,5-dienylidene)methyl)-1,2,4-triazine-3,5(2H,4H)-dione (2a-f), were obtained via reaction of substituted uridine (1) and different aromatic aldehydes separately. Synthesized NUDs were further characterized using FTIR, 1H & 13C-NMR, mass and element analysis data. Characterized NUDs were assessed for their inhibition potential against DENV 2. Synthesized NUDs were also evaluated for their cytotoxicity towards Vero cells by MTT assay method. This investigation successfully synthesized NUDs 2a-f and reported their high inhibitory activity against DENV 2. The synthesized NUDs exhibited negligible cytotoxicity. High anti-viral activity against DENV 2 serotype and least/no cytotoxicity of NUDs suggests their importance in the treatment of dengue. Present study recommends that in future these NUDs must be investigated for their clinical importance to establish them as a choice for dengue treatment.

The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD + synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.

Chikungunya virus (CHIKV) is an enveloped, positive-sense RNA virus that has re-emerged to cause millions of human infections worldwide. In humans, acute CHIKV infection causes fever and severe muscle and joint pain. Chronic and debilitating arthritis and joint pain can persist for months to years. To date, there are no approved antivirals against CHIKV. Recently, the ribonucleoside analog 4'-fluorouridine (4'-FlU) was reported as a highly potent orally available inhibitor of SARS-CoV-2, respiratory syncytial virus, and influenza virus replication. In this study, we assessed 4'-FlU's potency and breadth of inhibition against a panel of alphaviruses including CHIKV, and found that it broadly suppressed alphavirus production in cell culture. 4'-FlU acted on the viral RNA replication step, and the first 4 hours post-infection were the critical time for its antiviral effect. In vitro replication assays identified nsP4 as the target of inhibition. In vivo , treatment with 4'-FlU reduced disease signs, inflammatory responses, and viral tissue burden in mouse models of CHIKV and Mayaro virus infection. Treatment initiated at 2 hours post-infection was most effective; however, treatment initiated as late as 24-48 hours post-infection produced measurable antiviral effects in the CHIKV mouse model. 4'-FlU showed effective oral delivery in our mouse model and resulted in the accumulation of both 4'-FlU and its bioactive triphosphate form in tissues relevant to arthritogenic alphavirus pathogenesis. Together, our data indicate that 4'-FlU inhibits CHIKV infection in vitro and in vivo and is a promising oral therapeutic candidate against CHIKV infection.IMPORTANCEAlphaviruses including chikungunya virus (CHIKV) are mosquito-borne positive-strand RNA viruses that can cause various diseases in humans. Although compounds that inhibit CHIKV and other alphaviruses have been identified in vitro , there are no licensed antivirals against CHIKV. Here, we investigated a ribonucleoside analog, 4'-fluorouridine (4'-FlU), and demonstrated that it inhibited infectious virus production by several alphaviruses in vitro and reduced virus burden in mouse models of CHIKV and Mayaro virus infection. Our studies also indicated that 4'-FlU treatment reduced CHIKV-induced footpad swelling and reduced the production of pro-inflammatory cytokines. Inhibition in the mouse model correlated with effective oral delivery of 4'-FlU and accumulation of both 4'-FlU and its bioactive form in relevant tissues. In summary, 4'-FlU exhibits potential as a novel anti-alphavirus agent targeting the replication of viral RNA., Competing Interests: G.R.P. is a coinventor on patent WO 2019/1736002 covering the composition of matter and the use of EIDD-2749 and its analogs as an antiviral treatment. This study could affect his personal financial status. The other authors declare no conflict of interest.

Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Wavelength-shifting molecular beacons were prepared from L-DNA. The clickable anchor for the two dyes, Cy3 and Cy5, was 2'- O -propargyl-L-uridine and was synthesized from L-ribose. Four clickable molecular beacons were prepared and double-modified with the azide dyes by a combination of click chemistry on a solid support for Cy3 during DNA synthesis and postsynthetic click chemistry for Cy5 in solution. Cy3 and Cy5 successfully formed a FRET pair in the beacons, and the closed form (red fluorescence) and the open form (green fluorescence) can be distinguished by the two-color fluorescence readout. Two molecular beacons were identified to show the greatest fluorescence contrast in temperature-dependent fluorescence measurements. The stability of the L-configured molecular beacons was demonstrated after several heating and cooling cycles as well as in the cell lysate. In comparison, D-configured molecular beacons showed a rapid decrease of fluorescence contrast in the cell lysate, which is caused by the opening of the beacons, probably due to degradation. This was confirmed in cell experiments using confocal microscopy. The L-configured molecular beacons are potential intracellular thermometers for future applications.

Coronaviruses are a group of enveloped viruses with non-segmented, single-stranded, and positive-sense RNA genomes. It belongs to the 'Coronaviridae family', responsible for various diseases, including the common cold, SARS, and MERS. The COVID-19 pandemic, which began in March 2020, has affected 209 countries, infected over a million people, and claimed over 50,000 lives. Significant efforts have been made by repurposing several approved drugs including antiviral, to combat the COVID-19 pandemic. Molnupiravir is found to be the first orally acting efficacious drug to treat COVID-19 cases. It was approved for medical use in the UK in November 2021 and other countries, including USFDA, which granted approval an emergency use authorization (EUA) for treating adults with mild to moderate COVID-19 patients. Considering the importance of molnupiravir, the present review deals with its various synthetic strategies, pharmacokinetics, bio-efficacy, toxicity, and safety profiles. The comprehensive information along with critical analysis will be very handy for a wide range of audience including medicinal chemists in the arena of antiviral drug discovery especially anti-viral drugs against any variant of COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: To review the pharmacokinetic (PK)-pharmacodynamic (PD) profiles, disease setting, dosing, and safety of oral and parenteral hypomethylating agents (HMAs) for the treatment of myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML), and to provide a multidisciplinary perspective on treatment selection and educational needs relating to HMA use., Data Sources: Clinical and real-world data for parenteral decitabine and azacitidine and two oral HMAs: decitabine-cedazuridine (DEC-C) for MDS and azacitidine (CC-486) for AML maintenance therapy., Data Summary: Differences in the PK-PD profiles of oral and parenteral HMA formulations have implications for their potential toxicities and planned use. Oral DEC-C (decitabine 35 mg and cedazuridine 100 mg) has demonstrated equivalent systemic area under the concentration-time curve (AUC) exposure to a 5-day regimen of intravenous (IV) decitabine 20 mg/m 2 and showed no significant difference in PD. The AUC equivalence of oral DEC-C and IV decitabine means that these regimens can be treated interchangeably (but must not be substituted within a cycle). Oral azacitidine has a distinct PK-PD profile versus IV or subcutaneous azacitidine, and the formulations are not bioequivalent or interchangeable owing to differences in plasma time-course kinetics and exposures. Clinical trials are ongoing to evaluate oral HMA combinations and novel oral HMAs, such as NTX-301 and ASTX030., Conclusions: Treatment with oral HMAs has the potential to improve quality of life, treatment adherence, and disease outcomes versus parenteral HMAs. Better education of multidisciplinary teams on the factors affecting HMA treatment selection may help to improve treatment outcomes in patients with MDS or AML., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: JK-S is Chair of the Summit & Julie Shah Foundation; is a member of Clinica Latina Board and Columbus Free Clinic Board; and has received honoraria or support for attending meetings from Hematology/Oncology Pharmacy Association and Vizient. AMZ is a Leukemia and Lymphoma Society Scholar in Clinical Research; has received research funding (institutional) from Abbvie, Aprea, Astex, BeyondSpring, Boehringer-Ingelheim, Celgene/BMS, Foran, Geron, Incyte, Kura, Medimmune/AstraZeneca, Novartis, Pfizer, Shattuck Labs, and Takeda; has received honoraria from AbbVie, Agios, ALX Oncology, Amgen, Astellas, BioCryst, Boehringer-Ingelheim, Celgene/BMS, Chiesi, Daiichi Sankyo, Epizyme, Genentech, Geron, Gilead, Incyte, Ionis, Janssen, Jazz, Kura, Mendus, Notable, Novartis, Orum, Otsuka, Pfizer, Regeneron, Schrodinger, Seattle Genetics, Servier, Syndax, Syros, Taiho Oncology, Takeda, Tyme, and Zentalis; and has served on clinical trial committees for Abbvie, ALX Oncology, BioCryst, Celgene/BMS, Geron, Gilead, Kura, Novartis, and Syros. RH and LB have no relevant conflicts of interest.

Messenger RNA (mRNA) can be sequenced via indirect approaches such as Sanger sequencing and next generation sequencing (NGS), or direct approaches like bottom-up mass spectrometry (MS). Direct sequencing allows the confirmation of RNA modifications. However, the conventional bottom-up MS approach involves time-consuming in-solution digestions that require a large amount of sample, and can lead to the RNase contamination of the LC-MS system and column. Here, we describe a platform that enables online nucleotide mapping of mRNAs via the use of immobilized RNase cartridges and 2D-LC-MS instrumentation. The online approach was compared to conventional offline digestion protocols adapted from two published studies. For this purpose, five model mRNAs of varying lengths (996-4521 nucleotides) and chemistries (unmodified uridine vs 5-methoxyuridine (5moU)) were analyzed. The profiles and sequence coverages obtained after RNase T1 digestion were discussed. The online nucleotide mapping achieved comparable or slightly greater sequence coverage for the 5 mRNAs (5.8-51.5%) in comparison to offline approaches (3.7-50.4%). The sequence coverage was increased to 65.6-85.6 and 69.7-85.0% when accounting for the presence of nonunique digestion products generated by the RNase T1 and A, respectively. The online nucleotide mapping significantly reduced the digestion time (from 15 to <5 min), increased the signal intensity by more than 10-fold in comparison to offline approaches.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase ( ALS ) gene but no mutation in acetyl-CoA carboxylase ( ACCase ) and transport inhibitor response1 ( TIR1 ) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes ( CYP71C42 and CYP71D55 ), one GST gene ( GSTT2 ), two glycosyltransferase genes ( rhamnosyltransferase 1 and IAAGLU ), and two ABC transporter genes ( ABCG1 and ABCG25 ) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli .

Background: Thyroid cancer, originating in the neck's thyroid gland, encompasses various types. Genetic mutations, particularly in BRAF and RET genes are crucial in its development. This study investigates the association between BRAF (rs113488022) and RET (rs77709286) polymorphisms and thyroid cancer risk in the Khyber Pakhtunkhwa (KP) population., Methods: Blood samples from 100 thyroid cancer patients and 100 healthy controls were genotyped using ARMS-PCR followed by gel electrophoresis and statistical analysis., Results: Analysis revealed a significant association between the minor allele T of BRAF (rs113488022) and thyroid cancer risk (P = 0.0001). Both genotypes of BRAF (rs113488022) showed significant associations with thyroid cancer risk (AT; P = 0.0012 and TT; P = 0.045). Conversely, the minor allele G of RET (rs77709286) exhibited a non-significant association with thyroid cancer risk (P = 0.2614), and neither genotype showed significant associations (CG; P = 0.317, GG; P = 0.651). Demographic and clinical parameters analysis using SPSS showed a non-significant association between BRAF and RET variants and age group (P = 0.878 and P = 0.536), gender (P = 0.587 and P = 0.21), tumor size (P = 0.796 and P = 0.765), or tumor localization (P = 0.689 and P = 0.727)., Conclusion: In conclusion, this study emphasizes the significant association between BRAF polymorphism and thyroid cancer risk, while RET polymorphism showed a less pronounced impact. Further validation using larger and specific datasets is essential to establish conclusive results., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

This study accessed the potential antimalarial activity of triterpene glycoside of H. atra through targeting orotidine 5-monophosphate decarboxylase protein (PfOMPDC) in P. falciparum by molecular docking. Nine triterpene glycosides from H. atra extract modeled the structure by the Corina web server and interacted with PfOMPDC protein by using Hex 8.0.0. The docking results were visualized and analyzed by Discovery Studio version 21.1.1. 17-Hydroxyfuscocineroside B showed the lowest binding energy in PfOMPDC interaction, which was -1,098.13 kJ/mol. Holothurin A3, echinoside A, and fuscocineroside C showed low binding energy. Nine triterpene glycosides of H. atra performed interaction with PfOMPDC protein at the same region. Holothurin A1 posed interaction with PfOMPDC protein by 8 hydrogen bonds, 3 hydrophobic interactions, and 8 unfavorable bonds. Several residues were detected in the same active sites of other triterpene glycosides. Residue TYR111 was identified in all triterpene glycoside complexes, except holothurin A3 and calcigeroside B. In summary, the triterpene glycoside of H. atra is potentially a drug candidate for malaria therapeutic agents. In vitro and in vivo studies were required for further investigation., Competing Interests: No potential conflict of interest was reported by the authors., (Copyright © 2024 Prawesty Diah Utami et al.)

Background: We describe the first case of a pediatric patient with acute intermittent porphyria and severe chronic porphyric neuropathy treated with givosiran, a small-interfering RNA that drastically decreases delta-aminolevulinic acid production and reduces porphyric attacks' recurrence., Case Report: A 12-year-old male patient with refractory acute intermittent porphyria and severe porphyric neuropathy was followed prospectively for 12 months after givosiran initiation (subcutaneous, 2.5 mg/kg monthly). Serial neurological, structural, and resting-state functional magnetic resonance imaging (MRI) evaluations were performed, including clinical scales and neurophysiological tests. Delta-aminolevulinic acid urinary levels dropped drastically during treatment. In parallel, all the administered neurological rating scales and neurophysiological assessments showed improvement in all domains. Moreover, an improvement in central motor conduction parameters and resting-state functional connectivity in the sensory-motor network was noticed. At the end of the follow-up, the patient could walk unaided after using a wheelchair for 5 years., Conclusions: A clear beneficial effect of givosiran was demonstrated in our patient with both clinical and peripheral nerve neurophysiologic outcome measures. Moreover, we first reported a potential role of givosiran in recovering central motor network impairment in acute intermittent porphyria (AIP), which was previously unknown. This study provides Class IV evidence that givosiran improves chronic porphyric neuropathy.

Background: Hypomethylating agents combined with venetoclax are effective regimens in patients with acute myeloid leukaemia who are ineligible for intensive chemotherapy. Decitabine and cedazuridine (ASTX727) is an oral formulation of decitabine that achieves equivalent area-under-curve exposure to intravenous decitabine. We performed a single centre phase 2 study to evaluate the efficacy and safety of ASTX727 plus venetoclax., Methods: This study enrolled patients with newly diagnosed (frontline treatment group) acute myeloid leukaemia who were ineligible for intensive chemotherapy (aged ≥75 years, an Eastern Cooperative Oncology Group [ECOG] performance status of 2-3, or major comorbidities) or relapsed or refractory acute myeloid leukaemia. Being aged 18 years or older and having an ECOG performance status of 2 or less were requirements for the relapsed or refractory disease treatment cohort, without any limits in the number of previous lines of therapy. Treatment consisted of ASTX727 (cedazuridine 100 mg and decitabine 35 mg) orally for 5 days and venetoclax 400 mg orally for 21-28 days in 28-day cycles. The primary outcome was overall response rate of ASTX727 plus venetoclax. Living patients who have not completed cycle one were not evaluable for response. Safety was analysed in all patients who started treatment. This study was registered on ClinicalTrials.gov (NCT04746235) and is ongoing. The data cutoff date for this analysis was Sept 22, 2023., Findings: Between March 16, 2021, and Sept 18, 2023, 62 patients were enrolled (49 frontline and 13 relapsed or refractory) with a median age of 78 years (IQR 73-82). 36 (58%) were male; 53 (85%) were White, 4 (6%) Black, 2 (3%) Asian and 3 (5%) other or did not answer. 48 (77%) of 62 patients were European LeukemiaNet 2022 adverse risk, 24 (39%) had antecedent myelodysplastic syndromes, 12 (19%) had previously failed a hypomethylating agent, ten (16%) had therapy-related acute myeloid leukaemia, and 11 (18%) had TP53 mutations. The median follow-up time was 18·3 months (IQR 8·8-23·3). The overall response rate was 30 (64%) of 47 patients (95% CI 49-77) in frontline cohort and six (46%) of 13 patients (19-75) in relapsed or refractory cohort. The most common grade 3 or worse treatment-emergent adverse events were febrile neutropenia in 11 (18%) of 62 patients, pneumonia in eight (13%), respiratory failure in five (8%), bacteraemia in four (6%), and sepsis in four (6%). Three deaths occurred in patients in remission (one sepsis, one gastrointestinal haemorrhage, and one respiratory failure) and were potentially treatment related., Interpretation: ASTX727 plus venetoclax is an active fully oral regimen and safe in most older or unfit patients with acute myeloid leukaemia. Our findings should be confirmed in larger multicentric studies., Funding: MD Anderson Cancer Center Support Grant, Myelodysplastic Syndrome/Acute Myeloid Leukaemia Moon Shot, Leukemia SPORE, Taiho Oncology, and Astex Pharmaceuticals., Competing Interests: Declaration of interests GG-M declares grants from Astex, Novartis, AbbVie, Genentech, Aprea, Curis, and Gilead; consulting fees from Astex, Acceleron, and BMS; and payment or honoraria from Astex, Acceleron, AbbVie, Gilead, Curis, Genentech, and BMS. NS declares research funding from Takeda Oncology, Astellas Pharma, Xencor, Stemline Therapeutics, and NextCure; consulting fees from Pfizer, GlaxoSmithKline, NKARTA, Autolus, and Sanofi; and payment or honoraria from Adaptive Biotechnologies, Novartis, Amgen, Takeda Oncology, Pfizer, Astellas Pharma, Sanofi, and BeiGene. GI declares grants from Celgene, Kura Oncology, Syndax, Merck, Cullinan, and Novartis and consulting fees from Novartis, Kura Oncology, and Nuprobe. NJ declares research funding from Pharmacyclics, AbbVie, Genentech, AstraZeneca, BMS, Pfizer, ADC Therapeutics, Cellectis, Adaptive Biotechnologies, Precision Biosciences, Fate Therapeutics, Kite and Gilead, Mingsight, Takeda, Medisix, Loxo Oncology, Novalgen, Dialectic Therapeutics, Newave, TransThera Sciences, Novartis, Carna Biosciences, Sana Biotechnology, and Kisoji Biotechnology and honoraria or advisory board participation from Pharmacyclics, Janssen, AbbVie, Genentech, AstraZeneca, BMS, Adaptive Biotechnologies, Kite and Gilead, Precision Biosciences, Beigene, Cellectis, MEI Pharma, Ipsen, CareDX, MingSight, and Novalgen. AM declares research funding from Lin Biosciences and Chimeric Therapeutics and travel support from Cero Bio. EJ declares grants from AbbVie, Adaptive Biotechnologies, Amgen, Pfizer, and Takeda and consulting fees from AbbVie, Adaptive Biotechnologies, Amgen, BMS, Genentech, Incyte, Novartis, Pfizer, and Takeda. CDD declares grants from AbbVie, Astex, ImmuneOnc, BMS, Cleave, Foghorn, Loxo, Rigel, and Servier; consulting fees from Amgen, AbbVie, Astellas, BMS, Genmab, GSK, Gilead, Jazz, Shrodinger, Servier, and Stemline; payment or honoraria from AbbVie, Astellas, BMS, Jazz, and Servier; travel support from Servier; and participation on a data safety board for Genmab. TK declares grants from BMS, AbbVie, Amgen, Ascentage Pharma Group, Astellas Pharma, DrenBio, Astex, AstraZeneca, BMS, Celgene, Incyte, Cellenkos, Cyclacel, Delta-Fly Pharma, Genentech, Genfleet, Glycomimetics, Iterion, Janssen, Jazz Pharmaceuticals, Pfizer, Pulmotect, Regeneron, and SELLAS; consulting fees from AbbVie, Agios, Daiichi Sankyo, Genentech, Genzyme, Jazz Pharmaceuticals, Liberum, Novartis, Pfizer, PinotBio, Pulmotect, Sanofi-Aventis, and Servier; and payment or honoraria from AbbVie, Agios, Daiichi Sankyo, DAVA Oncology, Delta-Fly, DrenBio, Genentech, Genfleet, Genzyme, Jazz Pharmaceuticals, Liberum, Novartis, Pfizer, Rigel, Sanofi-Aventis, SELLAS, and Servier. ND declares grants from Daiichi-Sankyo, BMS, Pfizer, Gilead, Servier, Genentech, Astellas, AbbVie, ImmunoGen, Amgen, Trillium, Hanmi, Trovagene, FATE Therapeutics, Novimmune, Glycomimetics, and KITE and consulting fees from Daiichi-Sankyo, BMS, Pfizer, Gilead, Servier, Genentech, Astellas, AbbVie, ImmunoGen, Amgen, Trillium, Arog, Novartis, Jazz, Celgene, Syndax, Shattuck Labs, Agios, KITE, and Stemline and Menarini. MK declares grants from AbbVie, Genentech, Gilead, ImmunoGen, MEI Pharma, Precision Biosciences, Rafael Pharmaceutical, Sanofi-Aventis, and Stemline Therapeutics; consulting fees from AbbVie, AstraZeneca, Boehringer, Hoffman-La Roche, Genentech, Gilead, Janssen, Legend Biotech, MEI Pharma, Redona, Sanofi-Aventis, Sellas, Stemline Therapeutics, and Vincerx; participation on an advisory board for AbbVie, Auxenion, GmbH, Bakx Therapeutics, Dark Blue Therapeutics, Hoffman-La Roche, Genentech, Gilead, Stemline Therapeutics, and Vincerx; has a board of directors role for Immune Oncology; clinical trial support from AbbVie, AstraZeneca, Cellectis, Genentech, Janssen, Pfizer, Sanofi-Aventis, and Stemline Therapeutics; and intellectual property from Reata Pharmaceuticals. HK declares grants from AbbVie, Amgen, Ascentage, BMS, Daiichi-Sankyo, Immunogen, Jazz, and Novartis and payment or honoraria from AbbVie, Amgen, Amphista, Ascentage, Astellas, Biologix, Curis, Ipsen Biopharmaceuticals, KAHR Medical, Labcorp, Novartis, Pfizer, Shenzhen Target Rx, Stemline, and Takeda. FR declares clinical trial support from Astex and Taiho Oncology and payment or honoraria from Taiho Oncology. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

5-Fluorouracil is now routinely used in chemo- and radiotherapy. Incorporated within DNA, the molecule is bound to the sugar backbone, forming the 5-fluorouridine sub-unit investigated in the present work. For the clinical usage of the latter, no information exists on the mechanisms that control the radiosensitizing effect at the molecular level. As low energy (< 12 eV) electrons are abundantly produced along the radiation tracks during cancer treatment using beams of high energy particles, we study how these ballistic secondary electrons damage the sensitizing molecule. The salient result from our study shows that the N -glycosidic bonds are principally affected with a cross-section of approximately two orders of magnitude higher than the canonical thymidine, reflecting to some degree the surviving factor of radiation-treated carcinoma cells with and without 5-fluorouracil incorporation. This result may help in the comprehension of the radiosensitizing effect of the fluoro-substituted thymidine in DNA.

Background: Hypomethylating agents are approved in higher-riskmyelodysplastic syndromes. The combination of a hypomethylating agent with venetoclax is standard of care in acute myeloid leukaemia. We investigated the safety and activity of the first totally oral combination of decitabine plus cedazuridine and venetoclax in patients with higher-risk-myelodysplastic syndromes and chronic myelomonocytic leukaemia., Methods: We did a single-centre, dose-escalation and dose-expansion, phase 1/2, clinical trial. Patients with treatment-naive higher-risk-myelodysplastic syndromes or chronic myelomonocytic leukaemia (risk level categorised as intermediate-2 or higher by the International Prognostic Scoring System) with excess blasts (>5%). Treatment consisted of oral decitabine 35 mg plus cedazuridine 100 mg on days 1-5 and venetoclax (variable doses of 100-400 mg, day 1 to 14, 28-day cycle). The primary outcomes were safety for the phase 1 part and the overall response for the phase 2 part of the study. The trial is ongoing and this analysis was not prespecified. This study is registered with ClinicalTrials.gov, NCT04655755, and is currently enrolling participants., Findings: Between Jan 21, 2021, and Jan 20, 2023, we enrolled 39 patients (nine in phase 1 and 30 in phase 2). The median age was 71 years (range 27-94), 28 (72%) patients were male, and 11 (28%) were female. The maximum tolerated dose was not reached, and the recommended phase 2 dose was established as oral decitabine 35 mg plus cedazuridine 100 mg for 5 days and venetoclax (400 mg) for 14 days. The most common grade 3-4 adverse events were thrombocytopenia (33 [85%] of 39), neutropenia (29 [74%]), and febrile neutropenia (eight [21%]). Four non-treatment-related deaths occurred on the study drugs due to sepsis (n=2), lung infection (n=1), and undetermined cause (n=1). The median follow-up time was 10·8 months (IQR 5·6-16·4). The overall response rate was 95% (95% CI 83-99; 37/39). 19 (49%) patients proceeded to hematopoietic stem-cell transplantation., Interpretation: This early analysis suggests that the combination of oral decitabine plus cedazuridine with venetoclax for higher-risk-myelodysplastic syndromes and chronic myelomonocytic leukaemia is safe in most patients, with encouraging activity. Longer follow-up will be needed to confirm these data., Funding: MD Anderson Cancer Center, MDS/AML Moon Shot, Genentech/AbbVie, and Astex Pharmaceuticals., Competing Interests: Declaration of interests GM-B declares research support from Takeda, Rigel, and IFN Therapeutics. KSC declares research funding from Amgen and consultancy fees from AbbVie. JI declares funding from the US National Institutes of Health, the Leukemia Research Foundation, and the US Department of Defense, and honoraria from AstraZeneca. KS declares consultancy fees from Novartis and honoraria from Otsuka Pharmaceuticals and Amgen. NS declares grants from Takeda Oncology, Astellas Pharma, Xencor, Stemline Therapeutics, and Nexcure; consultancy fees from Pfizer, GSK, NKARTA, and Sanofi; and honoraria from Adaptive Biotechologies, Novartis, Amgen, Takeda, Pfizer, Astellas Pharma, Sanofi, and BeiGene. GCI declares consultancy fees from Novartis, Kura Oncology, and Nuprobe; and research funding from Celgene, Kura Oncology, Syndax, Merck, Cullinan, and Novartis. ND declares grants from Daiichi-Sankyo, Bristol Myers Squibb (BMS), Pfizer, Gilead, Servier, Genentech, Astellas, AbbVie, ImmunoGen, Amgen, Trillium, Hanmi, Trovagene, FATE Therapeutics, Novimmune, Glycomimetics, and KITE; and consultancy fees from Daiichi-Sankyo, BMS, Pfizer, Gilead, Servier, Genentech, Astellas, AbbVie, ImmunoGen, Amgen, Trillium, Arog, Novartis, Jazz Pharmaceuticals, Celgene, Syndax, Shattuck Labs, Agios, KITE, and Stemline/Menarini. TMK declares grants from BMS, Abbvie, Amgen, Ascentage Pharma Group, Astellas Pharma, DrenBio, Astex, AstraZeneca, Celgene, Incyte, Cellenkos, Cyclacel, Delta-Fly Pharma, Genentech, Genfleet, Glycomimetics, Iterion, Janssen, Jazz Pharmaceuticals, Pfizer, Pulmotect, Regeneron, and SELLAS; consultancy fees from AbbVie, Agios, Daiichi Sankyo, Genentech, Genzyme, Jazz Pharmaceuticals, Liberum, Novartis, Pfizer, PinotBio, Pukmotect, Sanofi-Aventis, Servier; and honoraria from AbbVie, Agios, Daiichi Sankyo, DAVA Oncology, Delta-Fly, DrenBio, Genentech, Genfleet, Genzyme, Jazz Pharmaceuticals, Liberum, Novartis, Pfizer, Rigel, Sanofi-Aventis, SELLAS, and Servier. FR declares research funding from ASTEX and TAIHO. EJ received research grants from AbbVie, Adaptive Biotechnologies, Amgen, Pfizer, and Takeda; and consultancy fees from AbbVie, Adaptive Biotechnologies, Amgen, BMS, Genentech, Incyte, Novartis, Pfizer, and Takeda. HMK declares research funding from Abbvie, Amgen, Ascentage, BMS, Daiichi Sankyo, Immunogen, Jazz Pharmaceuticals, and Novartis; and honoraria from Abbvie, Amgen, Amphista, Ascentage, Astellas, Biologix, Curis, Ipsen Biopharmaceuticals, KAHR Medical, Labcorp, Novartis, Pfizer, Precision Biosciences, Shenzhen Target Rx, Stemline, and Takeda. GG-M declares research funding from Astex Pharmaceuticals, Novartis, Abbvie, BMS, Genentech, Aprea Therapeutics, Curis, and Gilead Sciences; consultancy fees from Astex Pharmaceuticals, Acceleron Pharma, and BMS; and honoraria from Astex Pharmaceuticals, Acceleron Pharma, Abbvie, Novartis, Gilead Sciences, Curis, Genentech, and BMS. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

While numerous Fabaceae seeds are a good nutritional source of high-quality protein, the use of some species is hampered by toxic effects caused by exposure to metabolites that accumulate in the seeds. One such species is the faba or broad bean ( Vicia faba L.), which accumulates vicine and convicine. These two glycoalkaloids cause favism, the breakdown of red blood cells in persons with a glucose-6-phosphate dehydrogenase deficiency. Because this is the most common enzyme deficiency worldwide, faba bean breeding efforts have focused on developing cultivars with low levels of these alkaloids. Consequently, quantification methods have been developed; however, they quantify vicine and convicine only and not the derivatives of these compounds that potentially generate the same bio-active molecules. Based on the recognition of previously unknown (con)vicine-containing compounds, we screened the fragmentation spectra of LC-MS/MS data from five faba bean cultivars using the characteristic fragments generated by (con)vicine. This resulted in the recognition of more than a hundred derivatives, of which 89 were tentatively identified. (Con)vicine was mainly derivatized through the addition of sugars, hydroxycinnamic acids, and dicarboxylic acids, with a group of compounds composed of two (con)vicine residues linked by dicarboxyl fatty acids. In general, the abundance profiles of the different derivatives in the five cultivars mimicked that of vicine and convicine, but some showed a derivative-specific profile. The description of the (con)vicine diversity will impact the interpretation of future studies on the biosynthesis of (con)vicine, and the content in potentially bio-active alkaloids in faba beans may be higher than that represented by the quantification of vicine and convicine alone.

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N 3 -methyluridine (2'-O-alkyl/2'-F-m 3 U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-m 3 U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT 20 with 2'-O-alkyl-m 3 U modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to m 3 U, 2'-F, 2'-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m 3 U and 2'-O-methyl-m 3 U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2'-O-propyl-m 3 U modification exhibits steric interactions with amino acid residues in the active site of 3'- and 5'-exonuclease, leading to enhanced stability. These combined data indicate that the 2'-modified-m 3 U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A simple, reliable, and efficient method for the gram-scale chemical synthesis of pyrimidine nucleosides functionalized with C5-carboxyl, nitrile, ester, amide, or amidine, starting from unprotected uridine and cytidine, is described. The protocol involves the synthesis of 5-trifluoromethyluridine and 5-trifluoromethylcytidine with Langlois reagent (CF 3 SO 2 Na) in the presence of tert-butyl hydroperoxide and subsequent transformation of the CF 3 group to the C5-C 'carbon substituents' under alkaline conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of 5-trifluoromethyluridine (5-CF 3 U) and 5-trifluoromethylcytidine (5-CF 3 C) Basic Protocol 2: Conversion of 5-CF 3 U and 5-CF 3 C to several C5-substituted ribonucleosides., (© 2024 Wiley Periodicals LLC.)

Objectives: To evaluate the efficacy and safety of molnupiravir for intra-household post-exposure prophylaxis (PEP) of COVID-19., Methods: MOVe-AHEAD was a randomized, controlled, double-blind, phase 3 trial comparing molnupiravir (800 mg twice daily for 5 days) with placebo. Eligible participants were adult, unvaccinated, asymptomatic household contacts of patients with laboratory-confirmed COVID-19. The primary efficacy endpoint was the incidence of COVID-19 through day 14 in modified intention-to-treat (MITT) participants (those who received ≥1 dose of study intervention) without detectable SARS-CoV-2 at baseline, termed the MITT-VN population. Superiority of molnupiravir was prespecified as a stratified one-sided p-value of <0.0249 for the treatment difference in this endpoint., Results: The MITT population comprised 763 participants randomized to molnupiravir and 764 to placebo; 83.6% had anti-SARS-CoV-2 antibodies at baseline. In the MITT-VN population, COVID-19 rates through day 14 were 6.5% with molnupiravir and 8.5% with placebo (one-sided p-value: 0.0848). In the molnupiravir arm, 25/35 of confirmed COVID-19 events (71.4%) occurred after completion of treatment (versus 17/49 [34.7%] for placebo). Adverse event rates were low and similar between molnupiravir and placebo., Conclusions: Molnupiravir was well-tolerated but did not meet the prespecified superiority criterion, possibly influenced in part by the high pre-existing immunity in the trial population., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sady A. Alpizar received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Jose Accini received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Duane C. Anderson received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Basem Eysa received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Isaí Medina-Piñón received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Norio Ohmagari received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Mykola M. Ostrovskyy received institutional research funding from Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Angela Aggrey-Amable was an employee of Merck & Co., Inc., Rahway, NJ, USA at the time this trial was conducted and owns stock in Merck & Co., Inc., Rahway, NJ, USA. Karen Beck, Dana Byrne, Staci Grayson, Peggy M. T. Hwang, Julia D. Lonchar, Julie Strizki, Yayun Xu, Amanda Paschke, Carisa S. De Anda, and Pamela S. Sears are employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, who may own stock and/or hold stock options in Merck & Co., Inc., Rahway, NJ, USA., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

5-methyluridine hemihydrate (5 mU) single crystals were synthesized by the slow solvent evaporation method. The physicochemical properties, such as frontier molecular orbitals, global reactivity indices and vibrational were computationally studied through density functional theory (DFT). In addition, structural, vibrational, and thermal properties were obtained by powder X-ray diffraction (PXRD), Raman spectroscopy, thermogravimetric (TG) analysis and differential scanning calorimetry (DSC). PXRD evaluated the structural behavior of 5 mU crystal in the temperature range of 300-460 K. The high-temperature PXRD results suggested that the crystal undergoes two dehydration processes, being a first occurring from the orthorhombic structure (P2 1 2 1 2) to triclinic (P1), in which the water losses occurred around 380 K. A second dehydration triggers the change from the triclinic structure to monoclinic (P2 1 ) within the 420-435 K temperature range. Furthermore, after this temperature, the anhydrous 5 mU suffers a melting process near 460 K, which is remarkably characterized as an irreversible process. Raman spectroscopy was carried out to identify the vibrational modes linked to the water molecule and the noticeable changes in these bands due to high-temperature effects around 380 K and 410 K. Indeed, changes on Raman bands, such as intensity inversion, the disappearance of bands associated with the hydrogen bonds formed from the water molecules and uracil group, and the ribose group were observed. Finally, this study provided details on the structural and vibrational changes caused by the dehydration of 5 mU crystals and the importance of hydrogen bonds for understanding the intermolecular interactions of the 5 mU, a methylated nucleoside with important biological functions., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A synthetic scheme was developed to derive a modified ribothymidine bearing a 3-(N-methylsulfamoyl)propyl group on 2'-oxygen (T MSP ). For synthesis initiation, a nucleophilic attack of 1,2-ethanediol on 5'-protected 2,2'-anhydro-ribothymidine was performed to selectively modify the 2'-position. After protection of the 3'-hydroxy group, the hydroxyethyl group was oxidized to the aldehyde, which was coupled with isobutyl (diethoxyphosphinyl)methanesulfonate through the Horner-Wadsworth-Emmons reaction to yield the sulfonate intermediate. The intermediate was further converted to the desired T MSP . Using the phosphoramidite units derived from nucleosides, we synthesized oligonucleotides incorporating T MSP . Oligonucleotides modified with T MSP were found to have duplex stability, resistance toward 3'-exonuclease digestion, and antisense activity comparable to that of the oligonucleotide modified with a previously reported 2'-O-methylcarbamoylethyl group. Based on these results and the generality of the synthetic scheme, 2'-O-sulfamoylalkyl modification is expected to be used for the modulation of the properties of oligonucleotides by changing the substituents on the nitrogen, enabling the oligonucleotides to possess suitable properties for antisense oligonucleotides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Penoxsulam (PNX) and silver nanoparticles (AgNPs) are likely to coexist in soils due to continuous use. However, the ecotoxicity of PNX in earthworms and the effect of AgNPs on PNX toxicity are unknown. Therefore, the toxicity of PNX (0.05, 0.5, and 2.5 mg/kg) single and co-exposure with AgNPs (10 mg/kg) after 28 and 56 days on Eisenia fetida (E. fetida) was investigated from biochemical, genetic, histopathological, and transcriptomic aspects. The results showed that the low concentration of PNX (0.05PNX) had almost no effect on the biochemical level of E. fetida. However, the addition of AgNPs resulted in 0.05PNX causing E. fetida to produce excessive reactive oxygen species, and the activity of antioxidant and detoxification enzymes were interfered, resulting in lipid peroxidation and DNA damage. From the genetic level, even the lowest concentration of PNX can significantly interfere with the expression of functional genes, thus inducing oxidative stress and apoptosis and inhibited reproductive behavior in E. fetida. The integrated biomarker response results at the biochemical and genetic levels showed that the comprehensive toxicity of PNX and PNX + AgNPs on E. fetida was PNX dose-dependent. And the toxicity of all co-exposure groups was greater than that of the PNX only exposure groups. Furthermore, the addition of AgNPs significantly increased the damage of PNX on E. fetida intestinal tissue. Meanwhile, transcriptomic analysis showed that PNX + AgNPs had a greater effect on E. fetida than PNX single, and multiple pathways related to oxidative stress, inflammation, and cellular process regulation were disturbed. These results provide a basis for comprehensive evaluation of the ecotoxicity of PNX and confirm that the AgNPs does increase the ecotoxicity of PNX in soil environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Penoxsulam is an important herbicide for the control of Echinochloa crus-galli (L.) P. Beauv. Two resistant populations 17GA (R1) and 16NXB (R2) showed 17- and 3-fold resistance to penoxsulam, respectively. A known resistance mutation of Trp-574-Leu in ALS gene and enhanced rates of penoxsulam metabolism likely involving GST contribute to penoxsulam resistance in R1 population. This population had resistance to the ALS-inhibitors pyribenzoxim and bispyribac‑sodium and the auxin herbicide quinclorac, but was susceptible to ACCase-inhibitors quizalofop-p-ethyl and cyhalofop-butyl. No known mutations in the ALS gene conferring target site resistance to ALS-inhibiting herbicides were presented in R2 population. However, penoxsulam metabolism in R2 plants was about 4-fold greater than in susceptible population 14YC (S0) plants. The enzyme inhibitors piperonyl butoxide, malathion and 4-chloro-7-nitrobenzoxadiazole reversed penoxsulam resistance in this population. GST and P450 enzyme activities and the genes of GST1-1, GST1-2, GST1-3, CYP81A18, CYP81A12, CYP81A21 were increased significantly in R2 population. These results indicate that multiple resistance mechanisms had occurred in E. crus-galli populations in central China and resistance needs to be managed effectively by diverse chemical and non-chemical methods., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The faba bean is one of the earliest domesticated crops, with both economic and environmental benefits. Like most legumes, faba beans are high in protein, and can be used to contribute to a balanced diet, or as a meat substitute. However, they also produce the anti-nutritional compounds, vicine and convicine (v-c), that when enzymatically degraded into reactive aglycones can potentially lead to hemolytic anemia or favism. Current methods of analysis use LC-UV, but are only suitable at high concentrations, and thus lack the selectivity and sensitivity to accurately quantitate the low-v-c genotypes currently being developed. We have developed and fully validated a rapid high-throughput LC-MS method for the analysis of v-c in faba beans by optimizing the extraction protocol and assessing the method of linearity, limit of detection, limit of quantitation, accuracy, precision and matrix effects. This method uses 10-times less starting material; removes the use of buffers, acids and organic chemicals; and improves precision and accuracy when compared to current methods., Competing Interests: The authors declare no conflict of interest.