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.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2\'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase β (Pol β), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol β incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3\'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG.

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P > 0.05). However, samples with 5 % DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5 %: 53.67 ± 22.01; 7 %: 33.92 ± 23.4; MF: 5 %: 44.5 ± 20.46; 7 %: 38.75 ± 27.4) (Data: mean ± SD; P > 0.05). Hence, thirty mares were inseminated: 15 with 5 % DMF and 15 with 7 % DMF. The pregnancy rate was 46 % (7/15) and 0 % (0/15) using the extender with 5 % or 7 % DMF, respectively (P = 0.003). To conclude, the use of 5 % or 7 % of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5 % DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.

Globalization and habitat destruction pose a significant threat to wildlife felids. Even though conservation banks for genetic materials have been created, the sperm cryopreservation with minimal cell damage is still a great challenge. Thus, this study aimed to compare the effects of two commercial extenders with different concentrations of alternative cryoprotectants on thawed sperm quality of domestic cats. Five adult cats were anaesthetized (using a combination of 40 μg/kg medetomidine associated to 5 mg/kg ketamine), and the semen was collected by electroejaculation (electrical stimulation of 2-3 V). Semen samples were evaluated for sperm characteristics (kinetics, morphology, membrane integrity and morphometry). Subsequently, they were sorted into two aliquots and centrifuged. The aliquots were added to a commercial extender containing 3% glycerol and 2% methylformamide (extender I) or 2% glycerol and 3% methylformamide (extender II), frozen, thawed (37°C/30 s) and reevaluated. Comparatively, the sperm kinetics and membrane integrity of fresh semen were higher (p < .002) than frozen samples in extender I and II. Total and progressive motility were lowest in the thawed samples. However, the subjective analysis indicated high sperm motility, since the kinetics evaluation was impaired by the low cell number in the thawed samples. There were no differences in sperm morphology between the groups. In the sperm morphometric analysis, a significant difference (p = .04) was identified in the length of the intermediate piece in extender II samples compared with fresh and extender I. Thus, it can be concluded that although the concentrations tested did not maintain the kinetic parameters and membrane integrity of spermatozoa after thawing, the extender with a lower concentration of glycerol was less toxic for maintaining the midpiece length.

This review presents an analysis of formamide, focussing on its occurrence in nature, its functional roles, and its promising applications in the context of the bioeconomy. We discuss the utilization of formamide as an innovative nitrogen source achieved through metabolic engineering. These approaches underscore formamide\'s potential in supporting growth and production in biotechnological processes. Furthermore, our review illuminates formamide\'s role as a nitrogen source capable of safeguarding cultivation systems against contamination in non-sterile conditions. This attribute adds an extra layer of practicality to its application, rendering it an attractive candidate for sustainable and resilient industrial practices. Additionally, the article unveils the versatility of formamide as a potential carbon source that could be combined with formate or CO2 assimilation pathways. However, its attributes, i.e., enriched nitrogen content and comparatively limited energy content, led to conclude that formamide is more suitable as a co-substrate and that its use as a sole source of carbon for biomass and bio-production is limited. Through our exploration of formamide\'s properties and its applications, this review underscores the significance of formamide as valuable resource for a large spectrum of industrial applications. KEY POINTS: • Formidases enable access to formamide as source of nitrogen, carbon, and energy • The formamide/formamidase system supports non-sterile fermentation • The nitrogen source formamide supports production of nitrogenous compounds.

Isomerization commonly occurs in synthetic cannabinoids (SCs). Owing to the few differences in their structure and properties, it is difficult to simultaneously separate and identify them. Thus, the identification of synthetic cannabinoids is challenging, posing a threat to public security. This study aims to separate and identify four SCs, which are 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3,3-dimethylbutyrate methyl ester (5F-MDMB-PICA), 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3-methylbutyrate ethyl ester (5F-EMB-PICA), N-(1-amino-2,2-dimethyl-1-oxobutyl-2-yl)-1-butyl-1H-indazole-3-formamide (ADB-BINACA), N-(1-carbamoyl-2-methylpropyl)-1-pentyl indazole-3-formamide (AB-PINACA).Supercritical fluid chromatography-mass spectroscopy (SFC-MS) can realize the effective separation of some cannabinoid isomers. However, most laboratories are not equipped with SFC-MS systems. Ultra-high performance liquid chromatography-high resolution mass spectroscopy (UHPLC-HRMS) effectively combines the excellent efficient separation characteristics of liquid chromatography and the powerful qualitative ability of mass spectrometry. It is a commonly used technical method for the detection of amide synthetic cannabinoids and their metabolites in vivo and in vitro because of its advantages of high accuracy and efficiency. Liquid chromatography allows the separation of tested components by exploiting the difference in the partition coefficients between the mobile and stationary phases. When the two phases are in relative motion, the tested components are divided between the two phases, facilitating the separation and analysis of each component. Although the difference in the polarities of the tested amide synthetic cannabinoid isomeric substances is extremely small, liquid chromatography can induce a strong separation effect. The advantages of UHPLC-HRMS include high resolution imparted by mass spectrometry and high sensitivity, allowing its application in the qualitative analysis of various substances. Through UHPLC-HRMS, trace analytes at the nanogram scale as well as pure drugs and their metabolites in biosamples can be detected. This study proposed a method for the determination of two pairs of amide synthetic cannabinoid isomers-5F-EMB-PICA and 5F-MDMB-PICA, ADB-BINACA and AB-PINACA-through UHPLC-HRMS. A Hypersil GOLD C18 column (100 mm×2.1 mm, 1.9 μm) was selected for separation via liquid chromatography, and gradient elution was performed with methanol containing 0.1% formic acid and a 0.1% formic acid aqueous solution containing 10 mmol/L ammonium formate. Full scan/data-dependent secondary mass spectrometry (Full MS/dd-MS2) was conducted in the positive ion mode for detection. The results indicated that the four synthetic cannabinoid isomers could be accurately detected under the abovementioned conditions. The resolution between 5F-EMB-PICA and 5F-MDMB-PICA was 2.06, while that between ADB-BINACA and AB-PINACA was 1.22, indicating the effective separation and detection of both pairs. Furthermore, method validation was conducted to ensure the accuracy of the proposed method. The relationship of the four amide synthetic cannabinoid isomers exhibited excellent linearity. The correlation coefficients (R2) were >0.99. Moreover, the matrix effects of the four SCs in hair samples were between 88.67% and 111.76% and the recoveries were 96.23%-105.11%. The intra-day and inter-day precisions (RSDs) were <10%. The proposed method was used to identify the case materials. AB-PINACA was detected in a hair sample at a content of 0.73 μg/g. 5F-MDMB-PICA was detected in a tobacco sample at a content of 11.3 mg/g. The results indicate that the proposed method can be used for the examination of practical samples conducted by public security organizations. This study provides a reference method for the identification of synthetic cannabinoid isomers.

Deep eutectic solvents in the extraction of plant metabolites have found many advantages, such as low toxicity, biodegradability, low cost and ease of preparation over the conventional methods. This work aims to compare natural deep eutectic solvents in extraction and optimization of oleoresin from Ferula gummosa and determining its chemical and structure properties. Box-Behnken design was applied to optimize the extraction of oleoresin from Ferula gummosa using eutectic solvents. The variables of extraction were extraction time, temperature, and ratio of eutectic solvents. Six mixtures of eutectic solvents including choline chloride/urea, acetic acid, lactic acid, formic acid, formamide and glycerol at ratios of 2:1 and 3:1 were evaluated. The highest yields were obtained for choline chloride/formic acid, choline chloride/formamide. The quadratic regression equation was set up as a predictive model with an R2 value of 0.85. The optimum condition was 6 h, 40 °C, and ratio 12.5% (w/v). No significant difference was found between the predicted and experimental yield. The main components of the oleoresin were β-pinene (40.27%), cylcofenchen (11.93%) and α-pinene (7.53%) as characterized by gas chromatography-mass spectrometry. The chemical structure study by spectroscopy showed that no solvents remained in the oleoresin. Therefore, F. gummosa oleoresin can be explored as a novel promising natural pharmaceutical ingredient extracted with eutectic solvents.

3\',5\'-Cyclic nucleotides play a fundamental role in modern biochemical processes and have been suggested to have played a central role at the origin of terrestrial life. In this work, we suggest that a formamide-based systems chemistry might account for their availability on the early Earth. In particular, we demonstrate that in a liquid formamide environment at elevated temperatures 3\',5\'-cyclic nucleotides are obtained in good yield and selectivity upon intramolecular cyclization of 5\'-phosphorylated nucleosides in the presence of carbodiimides.

The development of alternative clean energy carriers is a key challenge for our society. Carbon-based hydrogen storage materials are well-suited to undergo reversible (de)hydrogenation reactions and the development of catalysts for the individual process steps is crucial. In the current state, noble metal-based catalysts still dominate this field. Here, a system for partially reversible and carbon-neutral hydrogen storage and release is reported. It is based on the dual-functional roles of formamides and uses a small molecule Fe-pincer complex as the catalyst, showing good stability and reusability with high productivity. Starting from formamides, quantitative production of CO-free hydrogen is achieved at high selectivity ( > 99.9%). This system works at modest temperatures of 90 °C, which can be easily supplied by the waste heat from e.g., proton-exchange membrane fuel cells. Employing such system, we achieve >70% H2 evolution efficiency and >99% H2 selectivity in 10 charge-discharge cycles, avoiding undesired carbon emission between cycles.