The importance of m6A topology in chicken embryo mRNA: a precise mapping of m6A at the conserved chicken ß-actin zipcode.

N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken ß-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of ß-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.

Emergent SARS-CoV-2 variants: comparative replication dynamics and high sensitivity to thapsigargin.

The struggle to control the COVID-19 pandemic is made challenging by the emergence of virulent SARS-CoV-2 variants. To gain insight into their replication dynamics, emergent Alpha (A), Beta (B) and Delta (D) SARS-CoV-2 variants were assessed for their infection performance in single variant- and co-infections. The effectiveness of thapsigargin (TG), a recently discovered broad-spectrum antiviral, against these variants was also examined. Of the 3 viruses, the D variant exhibited the highest replication rate and was most able to spread to in-contact cells; its replication rate at 24 h post-infection (hpi) based on progeny viral RNA production was over 4 times that of variant A and 9 times more than the B variant. In co-infections, the D variant boosted the replication of its co-infected partners at the expense of its own initial performance. Furthermore, co-infection with AD or AB combination conferred replication synergy where total progeny (RNA) output was greater than the sum of corresponding single-variant infections. All variants were highly sensitive to TG inhibition. A single pre-infection priming dose of TG effectively blocked all single-variant infections and every combination (AB, AD, BD variants) of co-infection at greater than 95% (relative to controls) at 72 hpi. Likewise, TG was effective in inhibiting each variant in active preexisting infection. In conclusion, against the current backdrop of the dominant D variant that could be further complicated by co-infection synergy with new variants, the growing list of viruses susceptible to TG, a promising host-centric antiviral, now includes a spectrum of contemporary SARS-CoV-2 viruses.

Diazophosphonates: Effective Surrogates for Diazoalkanes in Pyrazole Synthesis.

Diazophosphonates, readily prepared from α-ketophosphonates by oxidation of the corresponding hydrazones in batch or in flow, are useful partners in 1,3-dipolar cycloaddition reactions to alkynes to give N-H pyrazoles, including the first intramolecular examples of such a process. The phosphoryl group imbues a number of desirable properties into the diazo 1,3-dipole. The electron-withdrawing nature of the phosphoryl stabilizes the diazo compound making it easier to handle, whilst the ability of the phosphoryl group to migrate readily in a [1,5]-sigmatropic rearrangement enables its transfer from C to N to aromatize the initial cycloadduct, and hence its facile removal from the final pyrazole product. Overall, the diazophosphonate acts as a surrogate for the much less stable diazoalkane in cycloadditions, with the phosphoryl group playing a vital, but traceless, role. The cycloaddition proceeds more readily with alkynes bearing electron-withdrawing groups, and is regiospecific with asymmetrical alkynes. The potential of diazophosphonates for use in bioorthogonal cycloadditions is demonstrated by their facile addition to strained alkynes.

Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry.

Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus, Respiratory Syncytial Virus and Influenza A Virus.

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG's antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.

CDK12 inhibition reduces abnormalities in cells from patients with myotonic dystrophy and in a mouse model.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

The Impact of Macrocycle Conformation on the Taxadiene-Forming Carbocation Cascade: Insight Gained from Sobralene, a Recently Discovered Verticillene Isomer.

DFT calculations on the carbocation intermediates that connect the biosynthetic pathways leading to the sand fly pheromone sobralene and taxadiene have been made. Establishment of the conformation of the macrocyclic carbocation intermediate required to produce the (Z)-C8,C9 alkene bond in sobralene has identified new conformations of the verticillyl carbocation intermediates on the taxadiene biosynthetic pathway. These "sobralene-like" carbocation conformations provide an exothermic pathway to taxadiene and are validated by comparison to closely related structures (X-ray and NMR).

Unusually high α-proton acidity of prolyl residues in cyclic peptides.

The acidity of the α-proton in peptides has an essential role in numerous biochemical reactions and underpins their stereochemical integrity, which is critical to their biological function. We report a detailed kinetic and computational study of the acidity of the α-proton in two cyclic peptide systems: diketopiperazine (DKP) and triketopiperazine (TKP). The kinetic acidity (protofugality) of the α-protons were determined though hydrogen deuterium exchange studies in aqueous solutions. The acidities of the α-proton in prolyl residues were increased by 3-89 fold relative to other amino acid residues (prolyl > glycyl ≫ alanyl > tyrosyl). Experimental and computational evidence for the stereoelectronic origins of this enhanced prolyl reactivity is presented. TKPs were 106-fold more reactive than their DKP analogues towards deprotonation, which we attribute to the advanced development of aromaticity in the earlier transition state for proton transfer in these cases. A Brønsted linear free energy analysis of the reaction data was conducted to provide estimates of α-proton pK as.

Atrial fibrillation in elderly patients with implantable loop recorders for unexplained syncope.

BACKGROUND: An implantable loop recorder (ILR) assists in the diagnosis of unexplained syncope and atrial fibrillation (AF). Both become prevalent with age. Limited data exist describing the incidence of AF as the diagnostic rhythm underlying syncope in the elderly. This study aims to report the incidence of AF in older adults with ILRs for unexplained syncope and identify clinical characteristics associated with AF in this population. METHODS: Retrospective observational study on patients with unexplained syncope seen in syncope clinics from two Canadian centers. Participants were ≥65 years old, without a history of AF, and received an ILR for unexplained syncope. Data were collected from patient's clinic charts. Arrhythmia diagnosis was based on ILR electrocardiogram reading during syncope (symptom-rhythm correlation). Fisher's exact test and the Student's t test were used to compare participants with and without AF. RESULTS: In our cohort of 222 patients, 124 were females and 98 were males. Mean age at implant was 80 ± 8 years. Arrhythmia was diagnosed in 98 patients (44.1%). Median time to diagnosis was 18 months. AF was diagnosed in 17 (7.7%) participants. There were fewer males in the AF group than the no AF group (11.8%, 46.8%, p = 0.01). Age, baseline EKG, and prevalence of hypertension, diabetes, stroke, or ischemic heart disease were not statistically different between patients with AF and without AF. CONCLUSIONS: Atrial fibrillation was a common diagnostic rhythm in this cohort of adults, aged 65 and older, with ILRs for unexplained syncope. It was observed more frequently in females.

Implantable loop recorders in the real world: a study of two Canadian centers.

PURPOSE: Implantable loop recorders (ILRs) are increasingly being used for ambulatory electrocardiography. We sought to evaluate ILR indications, diagnostic yield, ILR-guided interventions, and complications in two Canadian centers. METHODS: This was a retrospective study using electronic medical records to identify ILR implants at Queen's University and the University of Manitoba. Information was collected on patient characteristics, medications, indication for implant, results of prior investigations, diagnostic outcome, and subsequent management. RESULTS: A total of 540 patients were identified; 386 had completed monitoring at time of analysis. Forty patients were lost to follow-up. Indications were unexplained syncope 84.8%, palpitations 12.8%, and suspected atrial fibrillation 11.7%. For syncope, ILRs documented arrhythmia or conduction disorder in 46%. Most common conditions were asystole/sinus pause (22%), complete heart block (10.4%), and atrial fibrillation (AF) (6.9%). After ILR diagnosis, 39.9% of implanted patients received pacemaker/ICD and 2.7% underwent catheter ablation. For palpitations, ILRs documented arrhythmia or conduction disorder in 60.4%. Most common conditions were AVNRT, AF, complete heart block, and ventricular tachycardia. After diagnosis, 25% underwent catheter ablation and 22.9% received pacemaker/ICD. For suspected AF, AF was diagnosed in 40%. Complications were observed in 3.3% of implanted patients: implant site infection 1.5%, non-infectious implant site pain requiring device removal or pocket revision 1.5%, 0.2% hypertrophic scar, and 0.2% device malfunction. CONCLUSIONS: An ILR has excellent diagnostic yield for syncope, palpitations, and suspected AF, and a considerable proportion of patients undergo ILR-directed interventions following monitoring. ILR implantation is a low-risk procedure.

Total synthesis of (-)-aritasone via the ultra-high pressure hetero-Diels-Alder dimerisation of (-)-pinocarvone.

This paper describes a total synthesis of the terpene-derived natural product aritasone via the hetero-Diels-Alder [4 + 2] cyclodimerisation of pinocarvove, which represents the proposed biosyntheic route. The hetero-Diels-Alder dimerisation of pinocarvone did not proceed under standard conditions, and ultra-high pressure (19.9 kbar) was required. As it seems unlikely that these ultra-high pressures are accessible within a plant cell, we suggest that the original biosynthetic hypothesis be reconsidered, and alternatives are discussed.

C-H Insertion as a Key Step to Spiro-Oxetanes, Scaffolds for Drug Discovery.

A new route to spiro-oxetanes, potential scaffolds for drug discovery, is described. The route is based on the selective 1,4-C-H insertion reactions of metallocarbenes, generated from simple carbonyl precursors in flow or batch mode, to give spiro-ß-lactones that are rapidly converted into spiro-oxetanes. The three-dimensional and lead-like properties of spiro-oxetanes are illustrated by the conversion of the 1-oxa-7-azaspiro[3,5]nonane scaffold into a range of functionalized derivatives.

Extracellular matrix gene expression profiling using microfluidics for colorectal carcinoma stratification.

In cancer, biomarkers have many potential applications including generation of a differential diagnosis, prediction of response to treatment, and monitoring disease progression. Many molecular biomarkers have been put forward for different diseases but most of them do not possess the required specificity and sensitivity. A biomarker with a high sensitivity has a low specificity and vice versa. The inaccuracy of the biomarkers currently in use has led to a compelling need to identify more accurate markers with diagnostic and prognostic significance. The aim of the present study was to use a novel, droplet-based, microfluidic platform to evaluate the prognostic value of a panel of thirty-four genes that regulate the composition of extracellular matrices in colorectal carcinoma. Our method is a novel approach as it uses using continuous-flowing Polymerase Chain Reaction for the sensitive detection and accurate quantitation of gene expression. We identified a panel of relevant extracellular matrix genes whose expression levels were measured by real-time quantitative polymerase chain reaction using Taqman® reagents in twenty-four pairs of matched colorectal cancer tumour and associated normal tissue. Differential expression patterns occurred between the normal and malignant tissue and correlated with histopathological parameters and overall surgical staging. The findings demonstrate that a droplet-based microfluidic quantitative PCR system enables biomarker classification. It was further possible to sub-classify colorectal cancer based on extracellular matrix protein expressing groups which in turn correlated with prognosis.

Synthesis of triazole-linked morpholino oligonucleotides via Cu(I) catalysed cycloaddition.

Triazole-linked morpholino ((TL)MO) oligonucleic acids were synthesised using the Cu(I) catalysed (3 + 2) azide-alkyne cycloaddition (CuAAC) reaction. The modified DNA analogues were incorporated into 13-mer sequences via solid phase synthesis. UV melting experiments showed that the (TL)MO modification gives higher Tm values than the corresponding (TL)DNA modification.

Stereoselective Synthesis of Functionalized Pyrrolidines by the Diverted N-H Insertion Reaction of Metallocarbenes with ß-Aminoketone Derivatives.

A highly stereoselective route to functionalized pyrrolidines by the metal-catalyzed diverted N-H insertion of a range of diazocarbonyl compounds with ß-aminoketone derivatives is described. A number of catalysts (rhodium(II) carboxylate dimers, copper(I) triflate, and an iron(III) porphyrin) are shown to promote the process under mild conditions to give a wide range of highly substituted proline derivatives. The reaction starts as a metallocarbene N-H insertion but is diverted by an intermolecular aldol reaction.

Accessing low-oxidation state taxanes: is taxadiene-4(5)-epoxide on the taxol biosynthetic pathway?

We have shown for the first time that taxadiene (3) can be epoxidised in a regio- and diastereoselective manner to provide taxadiene-4(5)-epoxide (12) as a single diastereoisomer, and that this epoxide can be rearranged to give taxa-4(20),11(12)-dien-5α-ol (4). Furthermore, the epoxide 12 rearranges under acidic conditions to give taxa-4(20),11(12)-dien-5α-ol (4), the known bridged ether OCT (5) and the new oxacyclotaxane (OCT2) 15. Contrary to previous speculation, taxadiene-4(5)-epoxide (12) is susceptible to rearrangement when exposed to an ironIII porphyrin, and these observations justify consideration of epoxide 12 as a chemically competent intermediate on the taxol biosynthetic pathway.

Stereoselective Synthesis of Highly Substituted Tetrahydrofurans through Diverted Carbene O-H Insertion Reaction.

Copper- or rhodium-catalyzed reactions of diazocarbonyl compounds with ß-hydroxyketones give highly substituted tetrahydrofurans with excellent diastereoselectivity. Under mild conditions, the single-step process starts as a carbene O-H insertion reaction, but is diverted by an intramolecular aldol reaction.

Alkyl halide-free heteroatom alkylation and epoxidation facilitated by a recyclable polymer-supported oxidant for the in-flow preparation of diazo compounds.

Highly reactive metal carbenes, generated from simple ketones via diazo compounds, including diazo-amides and -phosphonates, using a recyclable reagent in-flow, are transient but versatile electrophiles for heteroatom alkylation reactions and for epoxide formation. The method produces no organic waste, with the only by-products being water, KI and nitrogen, without the attendant hazards of isolation of intermediate diazo compounds.

Microfluidic droplet-based PCR instrumentation for high-throughput gene expression profiling and biomarker discovery.

PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. Real-time quantitative PCR (RT-qPCR) has become a definitive technique for quantitating differences in gene expression levels between samples. Yet, in spite of this importance, reliable methods to quantitate nucleic acid amounts in a higher throughput remain elusive. In the following paper, a unique design to quantify gene expression levels at the nanoscale in a continuous flow system is presented. Fully automated, high-throughput, low volume amplification of deoxynucleotides (DNA) in a droplet based microfluidic system is described. Unlike some conventional qPCR instrumentation that use integrated fluidic circuits or plate arrays, the instrument performs qPCR in a continuous, micro-droplet flowing process with droplet generation, distinctive reagent mixing, thermal cycling and optical detection platforms all combined on one complete instrument. Detailed experimental profiling of reactions of less than 300 nl total volume is achieved using the platform demonstrating the dynamic range to be 4 order logs and consistent instrument sensitivity. Furthermore, reduced pipetting steps by as much as 90% and a unique degree of hands-free automation makes the analytical possibilities for this instrumentation far reaching. In conclusion, a discussion of the first demonstrations of this approach to perform novel, continuous high-throughput biological screens is presented. The results generated from the instrument, when compared with commercial instrumentation, demonstrate the instrument reliability and robustness to carry out further studies of clinical significance with added throughput and economic benefits.

High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.