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.

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.

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.

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.

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.

Nucleoside-modified AdoMet analogues for differential methyltransferase targeting.

Methyltransferases (MTases) modify a wide range of biomolecules using S-adenosyl-l-methionine (AdoMet) as the cosubstrate. Synthetic AdoMet analogues are powerful tools to site-specifically introduce a variety of functional groups and exhibit potential to be converted only by distinct MTases. Extending the size of the substituent at the sulfur/selenium atom provides selectivity among MTases but is insufficient to discriminate between promiscuous MTases. We present a panel of AdoMet analogues differing in the nucleoside moiety (NM-AdoMets). These NM-AdoMets were efficiently produced by a previously uncharacterized methionine adenosyltransferase (MAT) from methionine and ATP analogues, such as ITP and N6-propargyl-ATP. The N6-modification changed the relative activity of three representative MTases up to 13-fold resulting in discrimination of substrates for the methyl transfer and could also be combined with transfer of allyl and propargyl groups.

Effect of Employment Status on Physical Activity and Sedentary Behavior Long-Term Post-Bariatric Surgery.

INTRODUCTION: Inactivity and weight regain are serious problems post-bariatric surgery. Nearly half of waking time is spent at work, representing an opportunity to accumulate physical activity and help avoid weight regain. PURPOSE: The purpose of this study is to evaluate potential differences in physical activity and sedentary time by employment status post-bariatric surgery. METHODS: A total of 48 adults (employed (n = 19), unemployed (n = 29)) aged 50.7 ± 9.4 years, BMI = 34.4 ± 10.1 kg/m2, and 10 ± 3 years post-surgery participated. ActivPAL accelerometers measured transitions, steps, and sedentary time for 7 days. RESULTS: Participants worked on average 8.7 ± 1.8 h/day. Twenty-one percent of employed met step/day guidelines on work-days compared to 10% of unemployed. Employed persons transitioned from sitting-to-standing more on work-days (58.6 ± 17.8) than unemployed (45.0 ± 15.4). Employment status did not influence activity or sedentarism on weekend/non-working-days. CONCLUSION: Employment status may be associated with meaningful improvements in activity post-bariatric surgery.

[Unsuccessful pain treatment over 6 years of a thoracic radiculopathy caused by an unrecognized Tarlov cyst]. / 6 Jahre erfolglose Schmerztherapie bei thorakaler Radikulopathie durch eine unerkannte Tarlov-Zyste.

This article presents a case of unsuccessful pharmaceutical and invasive pain treatment for 6 years without any adequate diagnostics in a female suffering from unilateral thoracic radiculopathy (Th8, right) leading to severe disability and unemployment. The origin was an undetected Tarlov cyst. After resection of the cyst the pain and other complaints disappeared (follow up: 8 months) without need for further pain medication. This case underlines the necessity of adequate diagnostics ahead of long-term pain treatment. Thoracic Tarlov cysts are very uncommon but should be included in the differential diagnosis because curative treatment may be possible.

Effects of Neighborhood Walkability on Physical Activity and Sedentary Behavior Long-Term Post-Bariatric Surgery.

ᅟ: Chronic inactivity and weight regain are serious health concerns following bariatric surgery. Neighborhood walkability is associated with higher physical activity and lower obesity rates in normal weight populations. PURPOSE: Explore the influence of neighborhood walkability on physical activity and sedentarism among long-term post-bariatric surgery patients. METHODS: Fifty-eight adults aged 50.5 ± 9.1 years, with a BMI of 34.6 ± 9.7 kg/m2 having undergone surgery 9.8 ± 3.15 years earlier participated in this study. Participants were asked to wear an ActivPAL™ tri-axial accelerometer attached to their mid-thigh for 7-consecutive days, 24 hours/day. The sample was separated into those that live in Car-Dependent (n = 23), Somewhat Walkable (n = 14), Very Walkable (n = 16), and Walker's Paradise (n = 5) neighborhoods as defined using Walk Score®. ANCOVA was performed comparing Walk Score® categories on steps and sedentary time controlling for age and sex. RESULTS: Neighborhood walkability did not influence either daily steps (F (3, 54) = 0.921, p = 0.437) or sedentary time (F (3, 54) = 0.465, p = 0.708), Car-Dependent (6359 ± 2712 steps, 9.54 ± 2.46 hrs), Somewhat Walkable (6563 ± 2989 steps, 9.07 ± 2.70 hrs), Very Walkable (5261 ± 2255 steps, 9.97 ± 2.06 hrs), and Walker's Paradise (6901 ± 1877 steps, 10.14 ± 0.815 hrs). CONCLUSION: Walkability does not appear to affect sedentary time or physical activity long-term post-surgery. As the built-environment does not seem to influence activity, sedentarism, or obesity as it does with a normal weight population, work needs to be done to tailor physical activity programming after bariatric surgery.

[The use of ureteral stents and catheters in renal transplantation.] / Empleo de catéteres y stents ureterales en el trasplante renal.

Mayor urological complications, fistulae and stenosis, mainly affect the vesicoureteral anastomosis and present in the early post-transplant period. The systematic use of ureteral catheters keeps selecbeing controversial with many groups using them only selectively depending on the existence of pretransplant or intraoperative risk factors. METHODS: We performed a bibliographic review through automatized search in the Medline bibliographic database, as the main bibliographic source, and also in Clinical Key. The search strategy included the following terms: "stent" AND "kidney transplantation". RESULTS: The bibliographic search revealed the protective effect of the use of ureteral catheters in the transplant ureteroneocystostomy for both development of fistulae (RR 0.29, 0.12 to 0.74, p=0.009) and stenosis (RR 0.27, 0.09 to 0.81, p=0.02). The use of catheters in immunosuppressed patients was associated with significant increase of the incidence of post-transplant urinary tract infections (RR 1.49 IC 95% 1.04 to 2.15, p=0.03) that was prevented by antibiotic prophylaxis with cotrimoxazole directed against pneumocistis carinii. The rates of permeability of self-expandable metallic stents and extra-anatomic bypasses in the treatment of ureteral stenosis after renal transplantation in high surgical risk patients or after the failure of previous surgery, has varied from 50% to 100%, with a limited number of patients included. CONCLUSIONS: The use of ureteral catheters in the extravesical ureteroneocystostomy reduces the incidence of anastomotic complications. Surgery is the treatment of choice of post-transplant ureteral stenosis. The use of metallic stents and extra-anatomic bypasses should be limited to complex ureteral stenosis when primary therapy has failed, in high surgical risk patients or chronic graft dysfunction.

Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free Protein Synthesis.

Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvß3, αvß5, and α5ß1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291-300. ©2016 AACR.