Chemo-enzymatic production of base-modified ATP analogues for polyadenylation of RNA.

Base-modified adenosine-5'-triphosphate (ATP) analogues are highly sought after as building blocks for mRNAs and non-coding RNAs, for genetic code expansion or as inhibitors. Current synthetic strategies lack efficient and robust 5'-triphosphorylation of adenosine derivatives or rely on costly phosphorylation reagents. Here, we combine the efficient organic synthesis of base-modified AMP analogues with enzymatic phosphorylation by a promiscuous polyphosphate kinase 2 class III from an unclassified Erysipelotrichaceae bacterium (EbPPK2) to generate a panel of C2-, N6-, or C8-modified ATP analogues. These can be incorporated into RNA using template independent poly(A) polymerase. C2-halogenated ATP analogues were incorporated best, with incorporations of 300 to >1000 nucleotides forming hypermodified poly(A) tails.

Tritrichomonas muris sensitizes the intestinal epithelium to doxorubicin-induced apoptosis.

Doxorubicin (DXR) is a widely used chemotherapy drug that can induce severe intestinal mucositis. While the influence of gut bacteria on DXR-induced damage has been documented, the role of eukaryotic commensals remains unexplored. We discovered Tritrichomonas muris ( Tmu ) in one of our mouse colonies exhibiting abnormal tuft cell hyperplasia, prompting an investigation into its impact on DXR-induced intestinal injury. Mice from Tmu -colonized and Tmu -excluded facilities were injected with DXR, and tissue morphology and gene expression were evaluated at acute injury (6 h) and peak regeneration (120 h) phases. Contrary to previous reports, DXR did not significantly alter villus height, crypt depth, or crypt density in any mice. However, we did observe apoptosis, measured by cleaved caspase 3 (CC3) staining, in intestinal crypts at 6 h post-DXR that was significantly higher in mice colonized by Tmu . Interestingly, while DXR did not alter the expression of active and facultative intestinal stem cell (ISC) marker genes in control mice, it significantly reduced their expression in Tmu + mice. Tmu , but not DXR, is also associated with increased inflammation and expression of the type 2 cytokines IL-5 and IL-13. However, pre-treatment of intestinal organoids with these cytokines is not sufficient to drive elevated DXR-induced apoptosis. These findings highlight the significant influence of commensal microbiota, particularly eukaryotic organisms like Tmu , on intestinal biology and response to chemotherapy, underscoring the complexity of gut microbiota interactions in drug-induced mucositis. NEW & NOTEWORTHY: Our study found that the eukaryotic commensal Tritrichomonas muris ( Tmu ) significantly increases DXR-induced intestinal apoptosis in mice, despite no changes in tissue morphology. Tmu also reduces intestinal stem cell gene expression post-DXR injury, and elevates inflammation and type 2 cytokine expression in the absence of injury. In vitro organoid assays suggest that type 2 cytokines alone are insufficient to promote increased DXR-associated apoptosis. These findings emphasize the complex role of gut microbiota in drug-induced intestinal damage.

MePMe-seq: antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing.

Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are methyl groups, making S-adenosyl-L-methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m6A from 2'-O-methyladenosine (Am) and N1-methyladenosine (m1A) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.

Enzymatic Synthesis of l-Methionine Analogues and Application in a Methyltransferase Catalysed Alkylation Cascade.

Chemical modification of small molecules is a key step for the development of pharmaceuticals. S-adenosyl-l-methionine (SAM) analogues are used by methyltransferases (MTs) to transfer alkyl, allyl and benzyl moieties chemo-, stereo- and regioselectively onto nucleophilic substrates, enabling an enzymatic way for specific derivatisation of a wide range of molecules. l-Methionine analogues are required for the synthesis of SAM analogues. Most of these are not commercially available. In nature, O-acetyl-l-homoserine sulfhydrolases (OAHS) catalyse the synthesis of l-methionine from O-acetyl-l-homoserine or l-homocysteine, and methyl mercaptan. Here, we investigated the substrate scope of ScOAHS from Saccharomyces cerevisiae for the production of l-methionine analogues from l-homocysteine and organic thiols. The promiscuous enzyme was used to synthesise nine different l-methionine analogues with modifications on the thioether residue up to a conversion of 75 %. ScOAHS was combined with an established MT dependent three-enzyme alkylation cascade, allowing transfer of in total seven moieties onto two MT substrates. For ethylation, conversion was nearly doubled with the new four-enzyme cascade, indicating a beneficial effect of the in situ production of l-methionine analogues with ScOAHS.

Comparative S-adenosyl-L-methionine analogue generation for selective biocatalytic Friedel-Crafts alkylation.

Methyltransferases provide excellent specificity in late-stage alkylation of biomolecules. Their dependence on S-adenosyl-L-methionine (SAM) mandates efficient access to SAM analogues for biocatalytic applications. We directly compared halide methyltransferase (HMT) and methionine adenosyltransferase (MAT) to access SAM analogues and explored their utility in cascade reactions with NovO for regioselective, late-stage Friedel-Crafts alkylation of a coumarin. The HMT cascade efficiently provided SAM for methylation, while the MAT cascade also supplied high levels of SAM analogues for alkylation reactions.

Biomimetic S-Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Effectiveness of COVID-19 Vaccination on Reduction of Hospitalizations and Deaths in Elderly Patients in Rio Grande do Norte, Brazil.

Since the COVID-19 pandemic emerged, vaccination has been the core strategy to mitigate the spread of SARS-CoV-2 in humans. This paper analyzes the impact of COVID-19 vaccination on hospitalizations and deaths in the state of Rio Grande do Norte, Brazil. We analyzed data from 23,516 hospitalized COVID-19 patients diagnosed between April 2020 and August 2021. We excluded the data from patients hospitalized through direct occupancy, unknown outcomes, and unconfirmed COVID-19 cases, resulting in data from 12,635 patients cross-referenced with the immunization status during hospitalization. Our results indicated that administering at least one dose of the immunizers was sufficient to significantly reduce the occurrence of moderate and severe COVID-19 cases among patients under 59 years. Considering the partially or fully immunized patients, the mean age is similar between the analyzed groups, despite the occurrence of comorbidities and higher than that observed among not immunized patients. Thus, immunized patients present lower Unified Score for Prioritization (USP) levels when diagnosed with COVID-19. Our data suggest that COVID-19 vaccination significantly reduced the hospitalization and death of elderly patients (60+ years) after administration of at least one dose. Comorbidities do not change the mean age of moderate/severe COVID-19 cases and the days required for the hospitalization of these patients.

Enzymatic Generation of Double-Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases.

Methyltransferases (MTases) have become an important tool for site-specific alkylation and biomolecular labelling. In biocatalytic cascades with methionine adenosyltransferases (MATs), transfer of functional moieties has been realized starting from methionine analogues and ATP. However, the widespread use of S-adenosyl-l-methionine (AdoMet) and the abundance of MTases accepting sulfonium centre modifications limit selective modification in mixtures. AdoMet analogues with additional modifications at the nucleoside moiety bear potential for acceptance by specific MTases. Here, we explored the generation of double-modified AdoMets by an engineered Methanocaldococcus jannaschii MAT (PC-MjMAT), using 19 ATP analogues in combination with two methionine analogues. This substrate screening was extended to cascade reactions and to MTase competition assays. Our results show that MTase targeting selectivity can be improved by using bulky substituents at the N6 of adenine. The facile access to >10 new AdoMet analogues provides the groundwork for developing MAT-MTase cascades for orthogonal biomolecular labelling.

Defining Endocytic Pathways of Fucoidan-Coated PIBCA Nanoparticles from the Design of their Surface Architecture.

PURPOSE: This work investigated the endocytic pathways taken by poly(isobutylcyanoacrylate) (PIBCA) nanoparticles differing in their surface composition and architecture, assuming that this might determine their efficiency of intracellular drug delivery. METHODS: Nanoparticles (A0, A25, A100, R0, R25 ) were prepared by anionic or redox radical emulsion polymerization using mixtures of dextran and fucoidan (0, 25, 100 % in fucoidan). Cell uptake was evaluated by incubating J774A.1 macrophages with nanoparticles. Endocytic pathways were studied by incubating cells with endocytic pathway inhibitors (chlorpromazine, genistein, cytochalasin D, methyl-ß-cyclodextrin and nocodazole) and nanoparticle uptake was evaluated by flow cytometry and confocal microscopy. RESULTS: The fucoidan-coated PIBCA nanoparticles A25 were internalized 3-fold more efficiently than R25 due to the different architecture of the fucoidan chains presented on the surface. Different fucoidan density and architecture led to different internalization pathway preferred by the cells. Large A100 nanoparticles with surface was covered with fucoidan chains in a loop and train configuration were internalized the most efficiently, 47-fold compared with A0, and 3-fold compared with R0 and R25 through non-endocytic energy-independent pathways and reached the cell cytoplasm. CONCLUSION: Internalization pathways of PIBCA nanoparticles by J774A.1 macrophages could be determined by nanoparticle fucoidan surface composition and architecture. In turn, this influenced the extent of internalization and localization of accumulated nanoparticles within cells. The results are of interest for rationalizing the design of nanoparticles for potential cytoplamic drug delivery by controlling the nature of the nanoparticle surface.

Treatment Patterns and Clinical Outcomes of Chronic Urticaria: Two-year Follow-up Results from the Scandinavian AWARE Study.

The AWARE (A World-wide Antihistamine-Refractory chronic urticaria patient Evaluation) study investigated outcomes in patients with chronic urticaria refractory to H1-antihistamine. The objective of the current study was to analyse the effects of treatment on patients' symptoms and quality of life for a period of up to 2 years. Over the 2 years, there was clear improvement in the high rates of disease burden from baseline, as evidenced by lower scores for disease severity scales, better quality of life, and a decreasing rate of medical resource utilization. However, this is the result of treatment adherence to the guidelines in highly specialized Scandinavian urticaria centres, and has its basis in the relatively low treatment intensity and control at enrolment. There is a need for greater adherence to the treatment guidelines and better management of antihistamine-refractory chronic urticaria.

Visible-Light Removable Photocaging Groups Accepted by MjMAT Variant: Structural Basis and Compatibility with DNA and RNA Methyltransferases.

Methylation and demethylation of DNA, RNA and proteins constitutes a major regulatory mechanism in epigenetic processes. Investigations would benefit from the ability to install photo-cleavable groups at methyltransferase target sites that block interactions with reader proteins until removed by non-damaging light in the visible spectrum. Engineered methionine adenosyltransferases (MATs) have been exploited in cascade reactions with methyltransferases (MTases) to modify biomolecules with non-natural groups, including first evidence for accepting photo-cleavable groups. We show that an engineered MAT from Methanocaldococcus jannaschii (PC-MjMAT) is 308-fold more efficient at converting ortho-nitrobenzyl-(ONB)-homocysteine than the wildtype enzyme. PC-MjMAT is active over a broad range of temperatures and compatible with MTases from mesophilic organisms. We solved the crystal structures of wildtype and PC-MjMAT in complex with AdoONB and a red-shifted derivative thereof. These structures reveal that aromatic stacking interactions within the ligands are key to accommodating the photocaging groups in PC-MjMAT. The enlargement of the binding pocket eliminates steric clashes to enable AdoMet analogue binding. Importantly, PC-MjMAT exhibits remarkable activity on methionine analogues with red-shifted ONB-derivatives enabling photo-deprotection of modified DNA by visible light.

Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites.

The RNA methyltransferase (MTase) complex METTL3-METTL14 transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenosines within its consensus sequence, the DRACH motif (D=A, G, U; R=A, G; H=A, C, U). Interestingly, this MTase complex shows remarkable promiscuity regarding the cosubstrate. This can be exploited to install nonnatural modifications, like clickable or photocaging groups. Clickable groups are widely used for subsequent functionalization and open a broad range of possibilities for downstream applications. Here, we elaborate on click chemistry for coupling of RNA to biotin to enrich MTase targets via streptavidin-coated magnetic beads. Importantly, after clicking and coupling to beads the modification becomes sterically demanding and stalls reverse transcriptases, leading to termination adjacent to the MTase target site. Using radioactively labeled primers in the reverse transcription, the modified position can be precisely identified on a sequencing gel via phosphor imaging.

Multiresponsive hydrogels and organogels based on photocaged cysteine.

Here we present the readily accessible amino acid 4,5-dimethoxy-2-nitrobenzyl-l-cysteine (DNC), as an ultra-low molecular weight gelator (MW = 316 g mol-1). Sonication of DNC in water or organic solvents as well as pH adjustment in water trigger gelation. A diverse set of stimuli (UV irradiation, oxidation, heat or pH change) induce a gel-sol transition. Moreover, the photo-triggered gel-sol transition was used to obtain a controlled cysteine release from the hydrogel.

Molecular systematics and biogeographic history of the African climbing-mouse complex (Dendromus).

Climbing mice in the genus Dendromus (sensu lato) are widely distributed in Africa, south of the Saharan Desert. The 17 currently recognized species in the genus range from widespread taxa to single-mountain endemics, and there is considerable variation across species with respect to habitats occupied. These habitats range from arid grasslands and savannahs to sub-alpine and alpine vegetation. Using the most comprehensive geographic and genetic survey to date and after reviewing many type specimens, we assess the systematics and biogeography of Dendromus. Given the structure of our molecular phylogenetic hypotheses, in which we recover six major clades, we propose the recognition of three genera within the Dendromus group (sensu lato): in addition to Dendromus (26 lineages), we suggest the retention of Megadendromus (monotypic) and the resurrection of the genus Poemys (six lineages). From our model-based molecular phylogenetic results and morphological comparisons, we suggest that six formerly synonymized taxa should be resurrected, and we highlight 14 previously undescribed lineages. We also constructed time-calibrations on our phylogeny, and performed ancestral area reconstructions using BioGeoBEARS. Based on fossil evidence, Dendromus appears to have had a widespread African distribution dating back to the Late Miocene (8-10 Ma), and our basal ancestral area reconstruction (Ethiopians Highlands + Eastern African Mountains + Zambezian region) supports this. Divergence of the six major clades we recover (Poemys, Megadendromus and four within Dendromus) occurred prior to or at the Miocene-Pliocene boundary 5.3 Ma. Biogeographically, Megadendromus is restricted to the Ethiopian Highlands. The ancestral area for Poemys is reconstructed as the Zambezian region, with species distributions ranging from South Africa to Western Africa. The ancestral area for Dendromus is reconstructed as the Ethiopian Highlands, with the ancestral areas of the four major clades being reconstructed as Ethiopian Highlands, Albertine Rift, South Africa or Western Africa. None of the four Dendromus clades are reciprocally monophyletic with respect to distributional area.

Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions.

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 Šrevealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification ("writing") of plasmid DNA and light-triggered removal ("erasing").

A Rad51-independent pathway promotes single-strand template repair in gene editing.

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

The association of cigarette smoking and pathological response to neoadjuvant platinum-based chemotherapy in patients undergoing treatment for urinary bladder cancer - A prospective European multicenter observational study of the EAU Young Academic Urologists (YAU) urothelial carcinoma working group.

OBJECTIVE: To prospectively study the impact of smoking on pathological response to neoadjuvant chemotherapy (NAC) in patients undergoing radical cystectomy (RC) for urothelial carcinoma of the bladder (UCB). MATERIALS & METHODS: We collected standard clinicopathological variables, including smoking status (never, former, current) in patients undergoing NAC and RC for UCB at 12 European tertiary care centers between 12/2013-12/2015. Clinicopathological variables were compared according to smoking status. Multivariable logistic regression models were built to assess the association of smoking status and a) complete (no residual disease), b) partial (residual, non-muscle invasive disease), c) no pathological response (residual muscle invasive or lymph node positive disease). Kaplan-Meier and Cox regression analyses were employed to study the impact of response to NAC on survival. RESULTS AND LIMITATIONS: Our final cohort consisted of 167 NAC patients with a median follow-up of 15 months (interquartile range (IQR) 9-26 months) of whom 48 (29%), 69 (41%), and 50 (30%) where never, former, and current smokers, respectively. Smoking was significantly associated with advanced age (p = 0.013), worse ECOG performance status (p = 0.049), and decreased pathological response to NAC (p = 0.045). On multivariable logistic regression analyses, former and current smoking status was significantly associated with lower odds of complete pathological response (odds ratio (OR) 0.37, 95% confidence interval (CI) 0.16-0.87, p = 0.023, and OR 0.34, 95% CI 0.13-0.85, p = 0.021), while current smoking status was significantly associated with a greater likelihood of no pathological response (OR 2.49, 95% CI 1.02-6.06, p = 0.045). Response to NAC was confirmed as powerful predictor of survival. CONCLUSIONS: Smoking status is adversely associated with pathological response to NAC. Smokers should be informed about these adverse effects, counseled regarding smoking cessation, and possibly be considered for immunotherpeutics as they may be more effective in smokers.