Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.

Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.

Palladium-Catalyzed Enantioselective [4+2] Cycloaddition of 4-Vinylbenzodioxinones with Barbiturate-Derived Alkenes: Con-struction of Chiral Spirobarbiturate-Chromanes.

In this paper, Pd-catalyzed [4+2] decarboxylative cycloaddition of 4-vinylbenzodioxinones with barbiturate-derived alkenes has been developed, leading to various spirobarbiturate-chromane derivatives in high yields with excellent diastereo- and enantioselectivities. The scale-up reaction and further derivation of the product were demonstrated. A plausible reaction mechanism was also proposed.

Palladium-Catalyzed [5 + 4] Cycloaddition of 4-Vinyl-4-Butyrolactones with N-Tosyl Azadienes: Construction of Nine-Membered Ring.

In this paper, we reported the palladium-catalyzed formal [5 + 4] cycloaddition reactions between 4-vinyl-4-butyrolactones (VBLs) and azadienes. Under mild reaction conditions, a wide range of benzofuran-fused 9-membered heterocyclic compounds had been provided in moderate to excellent yields with exclusive regioselectivities and excellent diastereoselectivities. The practical applicability of the synthesis was demonstrated through scale-up reaction and further transformation.

Phosphine-Catalyzed (4 + 2) Annulation of Allenoates Bearing Acidic Hydrogen with 1,1-Dicyanoalkenes.

α-succinimide-substituted allenoates were employed as phosphine acceptors in phosphine-catalyzed (4 + 2) annulation with 1,1-dicyanoalkenes. They served as C4 synthons in the annulation reaction under mild reaction conditions and produced hexahydroisoindole derivatives in moderate to high yields with good to excellent diastereoselectivities.

Palladium-Catalyzed (4 + 1) Annulation of 4-Vinylbenzodioxinones with Sulfur Ylides: Diastereoselective Synthesis of Dihydrobenzofuran Derivatives.

Palladium-catalyzed (4 + 1) annulation of 4-vinylbenzodioxinones with sulfur ylides has been developed to afford various dihydrobenzofuran derivatives in moderate to high yields with excellent diastereoselectivities. The scale-up reaction and further derivations of the product worked well, demonstrating the application potential of the current reaction in organic synthesis.

BF3·OEt2 Catalyzed Cascade [4 + 2] Benzannulation of Vinyloxiranes with Coumarins to Construct Benzocoumarin Derivatives.

A BF3·OEt2-catalyzed cascade cyclization reaction of vinyloxirane with coumarin is described, affording the benzocoumarin derivatives with moderate to excellent yields (72-92%). The reaction demonstrates exceptional substrate tolerance and has been extensively explored for its potential in drug development, including scale-up experiments, functional group transformations, and screening of the products for anticancer activity. Moreover, the reaction mechanism has been rigorously validated through intermediate trapping and control experiments. Additionally, this reaction represents the uncommon nonmetal catalyzed intermolecular cyclization of vinyloxiranes.

Palladium-catalyzed [4 + 2] cycloaddition of 2-methylidenetrimethylene carbonate or methylene cyclic carbamate with sulfamate-derived cyclic imines.

A palladium-catalyzed [4 + 2] cycloaddition of 2-methylidenetrimethylene carbonate or methylene cyclic carbamate with sulfamate-derived cyclic imines has been successfully developed under mild reaction conditions, affording pharmacologically interesting oxazine or hydropyrimidine derivatives in high yields (up to 99% yield). Furthermore, the cycloaddition reactions could be efficiently scaled up and several synthetic transformations were accomplished for the construction of other useful 1,3-oxazine and hydropyrimidinone derivatives.

An 'on-off-on' fluorescent switch based on a luminous covalent organic framework for the rapid and selective detection of glyphosate.

Glyphosate, the most used herbicide in the world, has a residue problem that cannot be ignored. However, glyphosate itself does not have fluorescence emission and lacks the conditions for fluorescence detection. In this work, a rapid and selective fluorescence detection method of glyphosate was designed by an 'on-off-on' fluorescent switch based on a luminous covalent organic framework (L-COF). Only the fixed concentration of Fe3+ as an intermediate could trigger the fluorescent switch and no incubation step was required. The proposed method showed good accuracy with a correlation coefficient of 0.9978. The method's limits of detection and quantitation were 0.88 and 2.93 µmol/L, which were lower than the maximum allowable residue limits in some regulations. Environmental water samples and tomatoes were selected as actual samples to verify the application in a complex matrix. A satisfactory mean recovery from 87% to 106% was gained. Furthermore, Fe3+ could induce fluorescence quenching of L-COF through the photo-induced electron transfer (PET) effect, while the addition of glyphosate could block the PET effect to achieve detection. These results demonstrated the proposed method had abilities to detect glyphosate and broaden the application of L-COF.

Pd-Catalyzed [4 + 2] cycloaddition of methylene cyclic carbamates with dihydropyrazolone-derived alkenes: synthesis of spiropyrazolones.

In this paper, a palladium-catalyzed [4 + 2] cycloaddition of 5-methylene-1,3-oxazinan-2-ones with 4-arylidene-2,4-dihydro-3H-pyrazol-3-ones has been developed to produce spiropyrazolones in high yields with excellent diastereoselectivities in nearly all cases. The cycloaddition reaction was scaled-up without significant loss of yield, and its synthetic utility has been demonstrated by further transformations of the products. The reaction type of N-Ts cyclic carbamates under palladium catalysis was extended to include [4 + 2] cycloaddition for the first time.

Phosphine-Catalyzed [3 + 2] Annulation of Morita-Baylis-Hillman Carbonates with Isoxazole-Based Alkenes.

A phosphine-catalyzed [3 + 2] annulation of Morita-Baylis-Hillman (MBH) carbonates with 3-methyl-4-nitro-5-styrylisoxazoles has been developed to afford various multifunctional isoxazoles in moderate to good yields with moderate to excellent diastereoselectivities. With a spirocyclic chiral phosphine as the catalyst, up to 89% ee was obtained.

In situ detection of plasma exosomal microRNA for lung cancer diagnosis using duplex-specific nuclease and MoS2 nanosheets.

MicroRNAs (miRNAs) encapsulated in tumor-derived exosomes are becoming ideal biomarkers for the early diagnosis and prognosis of lung cancer. However, the accuracy and sensitivity are often hampered by the extraction process of exosomal miRNA using traditional methods. Herein, this study developed a fluorogenic quantitative detection method for exosomal miRNA using the fluorescence quenching properties of molybdenum disulfide (MoS2) nanosheets and the enzyme-assisted signal amplification properties of duplex-specific nuclease (DSN). First, a fluorescently-labeled nucleic acid probe was used to hybridize the target miRNA to form a DNA/RNA hybrid structure. Under the action of the DSN, the DNA single strand in the DNA/RNA hybrid strand was selectively digested into smaller oligonucleotide fragments. At the same time, the released miRNA target triggers the next reaction cycle, so as to achieve signal amplification. Then, MoS2 was used to selectively quench the fluorescence of the undigested probe leaving the fluorescent signal of the fluorescently-labeled probe fragments. The fluorometric signals for miRNA-21 had a maximum excitation/emission wavelength of 488/518 nm. Most importantly, the biosensor was then applied for the accurate quantitative detection of miRNA-21 in exosome lysates extracted from human plasma and this method was able to successfully distinguish lung cancer patients from healthy people. This biosensor provides a simple, rapid, and a highly specific quantitative method for exosomal miRNA and has promising potential to be used in the early diagnosis of lung cancer.

Palladium-catalyzed stereoselective (3 + 2) cycloaddition of vinylethylene carbonates with cyclic N-sulfonyl ketimines.

A diastereoselective (3 + 2) cycloaddition of N-sulfonyl ketimines with vinylethylene carbonates (VECs) in the presence of Pd2dba3·CHCl3 and PPh3 has been developed. The reaction of various substituted VECs and diverse cyclic N-sulfonyl ketimines proceeded smoothly under mild conditions, giving highly functionalized oxazolidine frameworks in good to excellent yields with moderate to good diastereoselectivities. With the use of spiroketal-based diphosphine SKP as a chiral ligand, an asymmetric version of the current (3 + 2) cycloaddition was achieved, and chiral products were obtained in >99% ee in most cases.

A simple "signal off-on" fluorescence nanoplatform for the label-free quantification of exosome-derived microRNA-21 in lung cancer plasma.

A simple nanoplatform based on molybdenum disulfide (MoS2) nanosheets, a fluorescence quencher (signal off), and a hybridization chain reaction (HCR) signal amplification (signal on) used for the enzyme-free, label-free, and low-background signal quantification of microRNA-21 in plasma exosome is reported. According to the sequence of microRNA-21, carboxy-fluorescein (FAM)-labeled hybridization probe 1 (FAM-H1) and hybridization probes 2 (FAM-H2) were designed with excitation maxima at 488 nm and emission maxima at 518 nm. MoS2 nanosheets could adsorb FAM-H1 and FAM-H2 and quenched their fluorescence signals to reduce the background signal. However, HCR was triggered when microRNA-21 was present. Consequently, HCR products containing a large number of FAM fluorophores can emit a strong fluorescence at 518 nm and could realize the detection of microRNA-21 as low as 6 pmol/L and had a wide linear relation of 0.01-25 nmol/L. This assay has the ability of single-base mismatch recognition and could identify microRNA-21 with high specificity. Most importantly, this approach was successfully applied to the detection of plasma exosomal microRNA-21 in patients with lung cancer, and it is proposed that other targets can also be detected by changing the FAM-H1 and FAM-H2 corresponding to the target sequence. Thus, a novel, hands-on strategy for liquid biopsy was proposed and has a potential application value in the early diagnosis of lung cancer.

Nanozyme Sensor Arrays Based on Heteroatom-Doped Graphene for Detecting Pesticides.

Pesticides, widely used for pest control and plant growth regulation, have posed a threat to the environment and human health. Conventional methods to analyze pesticide residues are not applied to resource-limited areas because of their high cost, complexity, and requirements for expensive instruments (such as GC/MS and LC/MS). To address these challenges, herein we fabricated colorimetric nanozyme sensor arrays based on heteroatom-doped graphene for detection of aromatic pesticides. The active sites of nanozymes could be differentially masked when different pesticides were adsorbed on the graphene, which in turn resulted in the decrease of their peroxidase-mimicking activities. On the basis of this principle, five pesticides (i.e., lactofen, fluoroxypyr-meptyl, bensulfuron-methyl, fomesafen, and diafenthiuron) from 5 to 500 µM were successfully discriminated by the sensor arrays. In addition, discrimination for different concentrations of each pesticide and different ratios of two mixed pesticides were also demonstrated. The practical application of the sensor arrays was further validated by successfully discriminating the pesticides in soil samples. This work not only provides a facile and cost-effective method to detect pesticides but also makes a positive contribution to food safety and environmental protection.

Cu-Catalysed synthesis of benzo[f]indole-2,4,9(3H)-triones by the reaction of 2-amino-1,4-napthoquinones with α-bromocarboxylates.

A copper-catalysed cascade ester amidation/radical cyclization of 2-amino-1,4-naphthoquinones with α-bromocarboxylates to afford benzo[f]indole-2,4,9(3H)-triones is described, and the reaction has a broad substrate scope and the desired products are obtained in mostly moderate to good yields. Mechanism-probing experiments indicate that the otherwise challenging radical coupling reaction of α-bromocarboxylates with 2-amino-1,4-naphthoquinones is facilitated by a 5-endo radical cyclization.

Phosphine Organocatalysis.

The hallmark of nucleophilic phosphine catalysis is the initial nucleophilic addition of a phosphine to an electrophilic starting material, producing a reactive zwitterionic intermediate, generally under mild conditions. In this Review, we classify nucleophilic phosphine catalysis reactions in terms of their electrophilic components. In the majority of cases, these electrophiles possess carbon-carbon multiple bonds: alkenes (section 2), allenes (section 3), alkynes (section 4), and Morita-Baylis-Hillman (MBH) alcohol derivatives (MBHADs; section 5). Within each of these sections, the reactions are compiled based on the nature of the second starting material-nucleophiles, dinucleophiles, electrophiles, and electrophile-nucleophiles. Nucleophilic phosphine catalysis reactions that occur via the initial addition to starting materials that do not possess carbon-carbon multiple bonds are collated in section 6. Although not catalytic in the phosphine, the formation of ylides through the nucleophilic addition of phosphines to carbon-carbon multiple bond-containing compounds is intimately related to the catalysis and is discussed in section 7. Finally, section 8 compiles miscellaneous topics, including annulations of the Hüisgen zwitterion, phosphine-mediated reductions, iminophosphorane organocatalysis, and catalytic variants of classical phosphine oxide-generating reactions.

RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome.

AIMS: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. METHODS AND RESULTS: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. CONCLUSION: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

Organocatalytic Asymmetric C(sp2 )-H Allylic Alkylation: Enantioselective Synthesis of Tetrasubstituted Allenoates.

Herein we describe the first organocatalytic asymmetric C(sp2 )-H allylation of racemic trisubstituted allenoates with Morita-Baylis-Hillman (MBH) carbonates to access axially chiral tetrasubstituted allenoates. Various trisubstituted allenoates and MBH carbonates were well tolerated under mild reaction conditions, providing novel chiral tetrasubstituted allenoates with adjacent axial chirality and tertiary carbon stereocenters in high yields with good to excellent diastereoselectivities and enantioselectivities.

Palladium-Catalyzed Asymmetric [4+2] Cycloaddition of 2-Methylidenetrimethylene Carbonate with Alkenes: Access to Chiral Tetrahydropyran-Fused Spirocyclic Scaffolds.

A palladium-catalyzed asymmetric [4+2] cycloaddition of 2-methylidenetrimethylene carbonate with alkenes derived from pyrazolones, indandione, or barbiturate has been successfully developed, affording pharmacologically interesting chiral tetrahydropyran-fused spirocyclic scaffolds. The target compounds were generated in good to excellent yields and with high enantioselectivity (up to 99 % ee). Furthermore, this cycloaddition reaction could be efficiently scaled up, and several synthetic transformations were accomplished for the construction of other useful chiral spiropyrazolone and spiroindandione derivatives.

Phosphine-Catalyzed [3+2] Annulation of ß-Sulfonamido-Substituted Enones with Sulfamate-Derived Cyclic Imines.

Phosphine-catalyzed [3+2] annulation of ß-sulfonamido-substituted enones and sulfamate-derived cyclic imines has been developed, giving a series of imidazoline derivatives in moderate to excellent yields with good to excellent diastereoselectivities. A scale-up reaction worked well under mild reaction conditions. A possible mechanism was proposed on the basis of the results obtained.