Site-Specific Incorporation of a Dithiolane Containing Amino Acid into Proteins.

We have genetically encoded a dithiolane containing amino acid (dtF) in Escherichia coli (E. coli) using a polyspecific aminoacyl-tRNA synthetase (aaRS)/amber suppressor tRNA pair. To demonstrate the utility of dtF for bioapplications, we synthesized gold nanoparticle (AuNP) constructs with a mutant superfolder green fluorescent protein (sfGFP) [sfGFP-AuNP] as a model for the protein-metal conjugation. The resulting sfGFP-AuNP constructs show directional homogeneity and enhanced chemical durability compared to their cysteine analogues toward excess environmental 1,4-dithiothreitol (DTT).

Rational design of a function-based selection method for genetically encoding acylated lysine derivatives.

A foundation of genetic code expansion is the evolution of orthogonal aminoacyl-tRNA synthetase to recognize a non-canonical amino acid, which typically involves read-through of amber stop codon in the genes used in selection. The process is time-consuming, labor-intensive, and susceptible to certain bias. As a complementation, herein, we rationally designed a function-based method to directed evolution of aminoacyl-tRNA synthetase for acylated lysine derivatives.

Progress toward a reduced phage genetic code.

All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to minimize the genetic code.

Small Molecules Targeting Mycobacterium tuberculosis Type II NADH Dehydrogenase Exhibit Antimycobacterial Activity.

The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90 nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.

Chemo- and Regioselective Organo-Photoredox Catalyzed Hydroformylation of Styrenes via a Radical Pathway.

An unprecedented, chemo- and regioselective, organo-photoredox catalyzed hydroformylation reaction of aryl olefins with diethoxyacetic acid as the formylation reagent is described. In contrast to traditional transition metal promoted ionic hydroformylation reactions, the new process follows a unique photoredox promoted, free radical pathway. In this process, a formyl radical equivalent, produced from diethoxacetic acid through a dye (4CzIPN) photocatalyzed, sequential oxidation-decarboxylation route, regio- and chemoselectively adds to a styrene substrate. Importantly, under the optimized reaction conditions the benzylic radical formed in this manner is reduced by SET from the anion radical of 4CzIPN to generate a benzylic anion. Finally, protonation produces the hydroformylation product. By using the new protocol, aldehydes can be generated regioselectively in up to 90% yield. A broad array of functional groups is tolerated in the process, which takes place under mild, metal-free conditions.

Numerical investigation of cavity self-oscillation and noise radiation induced by turbulent flow at non-zero inclination angle.

A hybrid numerical approach was used on a three-dimensional cavity at a non-zero inclination angle of the upstream section to reveal the mechanism of self-oscillation and the characteristics of far-field sound field. In this hybrid approach, the unsteady flow physics was captured by a compressible large eddy simulation, and the far-field sound field was calculated by the FW-H integral equation, with the noise source provided by near-field calculation. The mechanism of self-oscillation was revealed based on the instantaneous flow field structure and the pressure inside the cavity. The effects of the position of opening, inclination angle and neck thickness on the frequency and amplitude of the fluctuation pressure inside the cavity were examined. Results showed that the frequency and the amplitude were sensitive to the inclination angle but not to the position of the opening. Under varying neck thicknesses, the fundamental frequencies changed, but the amplitude remained almost constant. The influences of the boundary layer thickness on the amplitude of the fluctuation pressure was also investigated. Results revealed that the oscillation was suppressed once the boundary layer thickness reached a critical value. The findings could provide a reference for a quiet car with a low sunroof buffeting noise.

Visible-Light-Promoted Nickel- and Organic-Dye-Cocatalyzed Formylation Reaction of Aryl Halides and Triflates and Vinyl Bromides with Diethoxyacetic Acid as a Formyl Equivalent.

A simple formylation reaction of aryl halides, aryl triflates, and vinyl bromides under synergistic nickel- and organic-dye-mediated photoredox catalysis is reported. Distinct from widely used palladium-catalyzed formylation processes, this reaction proceeds by a two-step mechanistic sequence involving initial in situ generation of the diethoxymethyl radical from diethoxyacetic acid by a 4CzIPN-mediated photoredox reaction. The formyl-radical equivalent then undergoes nickel-catalyzed substitution reactions with aryl halides and triflates and vinyl bromides to form the corresponding aldehyde products. Significantly, besides aryl bromides, less reactive aryl chlorides and triflates and vinyl halides serve as effective substrates for this process. Since the mild conditions involved in this reaction tolerate a plethora of functional groups, the process can be applied to the efficient preparation of diverse aromatic aldehydes.

Synthesis of Aldehydes by Organocatalytic Formylation Reactions of Boronic Acids with Glyoxylic Acid.

Reported herein is a conceptually novel organocatalytic strategy for the formylation of boronic acids. New reactivity is engineered into the α-amino-acid-forming Petasis reaction occurring between aryl boronic acids, amines, and glyoxylic acids to prepare aldehydes. The operational simplicity of the process and its ability to generate structurally diverse and valued aryl, heteroaryl, and α,ß-unsaturated aldehydes containing a wide array of functional groups, demonstrates the practical utility of the new synthetic strategy.

Dynamic Kinetic Resolution of Biaryl Lactones via a Chiral Bifunctional Amine Thiourea-Catalyzed Highly Atropo-enantioselective Transesterification.

A solution to the unmet synthetic challenge of achieving highly atropo-enantioselective transesterification of Bringmann's lactones has been realized, employing a chiral bifunctional amine thiourea as promoter. The synergistic activation of the lactones and alcohols/phenols by the respective thiourea and amine groups is crucial for achieving the highly enantioselective, high-yielding dynamic kinetic resolution process. This protocol gives highly optically pure, axially chiral biaryl compounds with a broad substrate scope under mild reaction conditions.

Facile Installation of 2-Reverse Prenyl Functionality into Indoles by a Tandem N-Alkylation-Aza-Cope Rearrangement Reaction and Its Application in Synthesis.

An unprecedented tandem N-alkylation-ionic aza-Cope (or Claisen) rearrangement-hydrolysis reaction of readily available indolyl bromides with enamines is described. Due to the complicated nature of the two processes, an operationally simple N-alkylation and subsequent microwave-irradiated ionic aza-Cope rearrangement-hydrolysis process has been uncovered. The tandem reaction serves as a powerful approach to the preparation of synthetically and biologically important, but challenging, 2-reverse quaternary-centered prenylated indoles with high efficiency. Notably, unusual nonaromatic 3-methylene-2,3-dihydro-1H-indole architectures, instead of aromatic indoles, are produced. Furthermore, the aza-Cope rearrangement reaction proceeds highly regioselectively to give the quaternary-centered reverse prenyl functionality, which often produces a mixture of two regioisomers by reported methods. The synthetic value of the resulting nonaromatic 3-methylene-2,3-dihydro-1H-indole architectures has been demonstrated as versatile building blocks in the efficient synthesis of structurally diverse 2-reverse prenylated indoles, such as indolines, indole-fused sultams and lactams, and the natural product bruceolline D.

Aniline-Promoted Cyclization-Replacement Cascade Reactions of 2-Hydroxycinnamaldehydes with Various Carbonic Nucleophiles through In Situ Formed N,O-Acetals.

In this study, we report the harnessing of new reactivity of N,O-acetals in an aminocatalytic fashion for organic synthesis. Unlike widely used strategies requiring the use of acids and/or elevated temperatures, direct replacement of the amine component of the N,O-acetals by carbon-centered nucleophiles for C-C bond formation is realized under mild reaction conditions. Furthermore, without necessary preformation of the N,O-acetals, an amine-catalyzed in situ formation of N,O-acetals is developed. Coupling both reactions into a one-pot operation enables the achievement of a catalytic process. We demonstrate the employment of simple anilines as promoters for the cyclization-substitution cascade reactions of trans-2-hydroxycinnamaldehydes with various carbonic nucleophiles including indoles, pyrroles, naphthols, phenols, and silyl enol ethers. The process offers an alternative approach to structurally diverse, "privileged" 2-substituted 2H-chromenes. The synthetic power of the new process is furthermore shown by its application in a 2-step synthesis of the natural product candenatenin E and for the facile installation of 2-substituted 2H-chromene moieties into biologically active indoles.

Construction of chiral bridged tricyclic benzopyrans: enantioselective catalytic Diels-Alder reaction and a one-pot reduction/acid-catalyzed stereoselective cyclization.

An asymmetric two-step approach to chiral bridged tricyclic benzopyrans, core structures featured in various natural products, is described. In the synthesis, an unprecedented enantioselective catalytic decarboxylative Diels-Alder reaction is developed using readily available coumarin-3-carboxylic acids and aldehydes as reactants under mild reaction conditions. Notably, the decarboxylation-assisted release of the catalyst enables the process to proceed efficiently with high enantio- and diastereoselectivity. Furthermore, a one-pot procedure for either a LiAlH4 - or NaBH4 -mediated reduction with subsequent acid-catalyzed intramolecular cyclization of the Diels-Alder adducts was identified for the efficient formation of the chiral bridged tricyclic benzopyrans.

Causative role of a novel intronic indel variant in FBN1 and maternal germinal mosaicism in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is an autosomal dominant connective tissue disease with wide clinical heterogeneity, and mainly caused by pathogenic variants in fibrillin-1 (FBN1). METHODS: A Chinese 4-generation MFS pedigree with 16 family members was recruited and exome sequencing (ES) was performed in the proband. Transcript analysis (patient RNA and minigene assays) and in silico structural analysis were used to determine the pathogenicity of the variant. In addition, germline mosaicism in family member (Ι:1) was assessed using quantitative fluorescent polymerase chain reaction (QF-PCR) and short tandem repeat PCR (STR) analyses. RESULTS: Two cis-compound benign intronic variants of FBN1 (c.3464-4 A > G and c.3464-5G > A) were identified in the proband by ES. As a compound variant, c.3464-5_3464-4delGAinsAG was found to be pathogenic and co-segregated with MFS. RNA studies indicated that aberrant transcripts were found only in patients and mutant-type clones. The variant c.3464-5_3464-4delGAinsAG caused erroneous integration of a 3 bp sequence into intron 28 and resulted in the insertion of one amino acid in the protein sequence (p.Ile1154_Asp1155insAla). Structural analyses suggested that p.Ile1154_Asp1155insAla affected the protein's secondary structure by interfering with one disulfide bond between Cys1140 and Cys1153 and causing the extension of an anti-parallel ß sheet in the calcium-binding epidermal growth factor-like (cbEGF)13 domain. In addition, the asymptomatic family member Ι:1 was deduced to be a gonadal mosaic as assessed by inconsistent results of sequencing and STR analysis. CONCLUSIONS: To our knowledge, FBN1 c.3464-5_3464-4delGAinsAG is the first identified pathogenic intronic indel variant affecting non-canonical splice sites in this gene. Our study reinforces the importance of assessing the pathogenic role of intronic variants at the mRNA level, with structural analysis, and the occurrence of mosaicism.

Mechanistic Studies of Small Molecule Ligands Selective to RNA Single G Bulges.

Small-molecule RNA binders have emerged as an important pharmacological modality. A profound understanding of the ligand selectivity, binding mode, and influential factors governing ligand engagement with RNA targets is the foundation for rational ligand design. Here, we report a novel class of coumarin derivatives exhibiting selective binding affinity towards single G RNA bulges. Harnessing the computational power of all-atom Gaussian accelerated Molecular Dynamics (GaMD) simulations, we unveiled a rare minor groove binding mode of the ligand with a key interaction between the coumarin moiety and the G bulge. This predicted binding mode is consistent with results obtained from structure-activity-relationship (SAR) studies and transverse relaxation measurements by NMR spectroscopy. We further generated 444 molecular descriptors from 69 coumarin derivatives and identified key contributors to the binding events, such as charge state and planarity, by lasso (least absolute shrinkage and selection operator) regression. Strikingly, small structure perturbations on these key contributors, such as the addition of a methyl group that disrupts the planarity of the ligand resulted in > 100-fold reduction in the binding affinity. Our work deepened the understanding of RNA-small molecule interactions and integrated a new generalizable platform for the rational design of selective small-molecule RNA binders.

A novel HOIP frameshift variant alleviates NF-kappaB signalling and sensitizes cells to TNF-induced death.

BACKGROUND: HOIP is the catalytic subunit of the E3 ligase complex (linear ubiquitin chain assembly complex), which is able to generate linear ubiquitin chains. However, the role of rare HOIP functionally deficient variants remains unclear. The pathogenic mechanism and the relationship with immune deficiency phenotypes remain to be clarified. METHODS: Based on a next-generation sequencing panel of 270 genes, we identified a HOIP deletion variant that causes common variable immunodeficiency disease. Bioinformatics analysis and cell-based experiments were performed to study the molecular mechanism by which the variant causes immunodeficiency diseases. FINDINGS: A homozygous loss-of-function variant in HOIP was identified. The variant causes a frameshift and generates a premature termination codon in messenger RNA, resulting in a C-terminal truncated HOIP mutant, that is, the loss of the linear ubiquitin chain-specific catalytic domain. The truncated HOIP mutant has impaired E3 ligase function in linear ubiquitination, leading to the suppression of canonical NF-κB signalling and increased TNF-induced multiple forms of cell death. INTERPRETATION: The loss-of-function HOIP variant accounts for the immune deficiencies. The canonical NF-κB pathway and cell death are involved in the pathogenesis of the disease. FUNDING: This study was funded by the National Natural Science Foundation of China (No. 82270444 and 81501851). RESEARCH IN CONTEXT: Evidence before this study LUBAC is the only known linear ubiquitin chain assembly complex for which HOIP is an essential catalytic subunit. Three HOIP variants have now been identified in two immunodeficient patients and functionally characterised. However, there have been no reports on the pathogenicity of only catalytic domain deletion variants in humans, or the pathogenic mechanisms of catalytic domain deletion variants. Added value of this study We report the first case of an autosomal recessive homozygous deletion variant that results in deletion of the HOIP catalytic structural domain. We demonstrate that this variant is a loss-of-function variant using a heterologous expression system. The variant has impaired E3 ligase function. It can still bind to other subunits of LUBAC, but it fails to generate linear ubiquitin chains. We also explored the underlying mechanisms by which this variant leads to immunodeficiency. The variant attenuates the canonical NF-κB and MAPK signalling cascades and increases the sensitivity of TNFα-induced diverse cell death and activation of mitochondrial apoptosis pathways. These findings provide support for the treatment and drug development of patients with inborn errors of immunity in HOIP and related signalling pathways. Implications of all the available evidence First, this study expands the HOIP pathogenic variant database and phenotypic spectrum. Furthermore, studies on the biological functions of pathogenic variants in relation to the NF-κB signalling pathway and cell death provided new understanding into the genetic basis and pathogenesis of HOIP-deficient immune disease, indicating the necessity of HOIP and related signalling pathway variants as diagnostic targets in patients with similar genetic deficiency phenotypes..

Crosstalk between KIF1C and PRKAR1A in left atrial myxoma.

Cardiac myxoma (CM) is the most common benign cardiac tumor, and most CMs are left atrial myxomas (LAMs). Six variations of KIF1C, c.899 A > T, c.772 T > G, c.352 A > T, c.2895 C > T, c.3049 G > A, and c.*442_*443dup in left atrial myxoma tissues are identified by whole-exome sequencing (WES) and Sanger sequencing. RNA-seq and function experiments show the reduction of the expression of KIF1C and PRKAR1A caused by rare variations of KIF1C. KIF1C is observed to be located in the nucleus, bind to the promoter region of PRKAR1A, and regulate its transcription. Reduction of KIF1C decreases PRKAR1A expression and activates the PKA, which causes an increase in ERK1/2 phosphorylation and SRC-mediated STAT3 activation, a reduction of CDH1, TP53, CDKN1A, and BAX, and eventually promotes tumor formation both in vitro and in vivo. The results suggest that inhibition of KIF1C promotes the pathogenesis of LAM through positive feedback formed by the crosstalk between KIF1C and PRKAR1A.

Construction of Enantioenriched γ,γ-Disubstituted Butenolides Enabled by Chiral Amine and Lewis Acid Cascade Cocatalysis.

Herein we report a cascade cocatalysis strategy for the facile construction of chiral γ,γ-disubstituted butenolides. The synthetic manifold employs simple alkynoic acids instead of the preformed silyloxy furans or 5-substituted furan-2(3H)-ones. In situ formed 5-substituted furan-2(3H)-ones by AgNO3 or Ph3PAuCl/AgOTf catalyzed cyclization of alkynoic acids can smoothly engage in the subsequent chiral diphenylprolinol TMS-ether catalyzed Michael and Michael-aldol reactions. The cascade process serves as a general approach to chiral quaternary γ,γ-disubstituted butenolides.

Kidney stone disease and cardiovascular events: a study on bidirectional causality based on mendelian randomization.

BACKGROUND: Kidney stone disease (KSD) has been reported to be associated with several cardiovascular diseases. However, the causality between the conditions remains unknown. In the study, we performed a study on bidirectional causality by two-sample Mendelian randomization (MR) to investigate the causality between KSD and cardiovascular diseases including coronary atherosclerosis, hypertension, and cardiomyopathy. METHODS: In the recent study, we performed a bidirectional two-sample MR study using available genome-wide association summary data from the online database MRBASE. We identified genetic variants associated with KSD in one European population from UK Biobank (version 2, n=462,933). Two phenotypes of samples were chosen from the population to define our genetic instrumental variables: (I) samples with the phenotype of kidney stone/ureter stone/bladder stone (ukb-b-8297), and (II) samples with the phenotype of kidney stone surgery/lithotripsy (ukb-b-13537). For cardiovascular diseases, we picked up another independent European population from FinnGen Biobank (n=93,421). We selected the exposure and outcome SNPs and then performed the two-sample MR using R package. RESULTS: After bidirectional causality by two-sample MR, we verified that genetic predisposition to KSD could increase the risk of coronary atherosclerosis (OR: 4.45×1037; SE=±7.80×1014, P for MR-Egger =0.024) and cardiomyopathy (OR: 5.35×1013; SE=±7.18×106, P for IVW=0.045 for finn-a-I9_CARDMYO, and OR: 3.60×1025; SE=±3.26×1012, P for IVW=0.041 for finn-a-I9_CARDMYOOTH) when we used ukb-b-13537 as exposure group. Furthermore, hypertension could increase the risk of KSD (OR: 1.001; SE=±1.00, P for IVW=0.003) when we used ukb-b-8297 as exposure group, without detected pleiotropy bias (P>0.05). CONCLUSIONS: We confirmed KSD may trigger causal pathological processes including coronary atherosclerosis and cardiomyopathy. Furthermore, hypertension may causally affect KSD.

Case Report: A Novel Homozygous Mutation in MYF5 Due to Paternal Uniparental Isodisomy of Chromosome 12 in a Case of External Ophthalmoplegia With Rib and Vertebral Anomalies.

External ophthalmoplegia with rib and vertebral anomalies (EORVA) is characterized by congenital nonprogressive external ophthalmoplegia, ptosis, scoliosis, torticollis, vertebral, and rib anomalies, caused by homozygous mutations in the myogenic factor 5 gene (MYF5) located on chromosome 12q21.31. Uniparental disomy (UPD) is a rare inheritance of a pair of chromosomes originating from only one parent. This study describes a case of an 8-year-old boy with ptosis, scoliosis, and dysmorphic hypoplastic ribs with fusion anomalies. Trio-based exome sequencing (trio-ES) identified a novel homozygous mutation c.191delC (p.Ala64Valfs*33) in MYF5 in the proband, with the father being heterozygous and the mother wild-type, as verified by Sanger sequencing. UPD identified from trio-ES variant call format data suggested the possibility of paternal UPD of chromosome 12 (UPD12pat) in the proband, further confirmed to be a complete isodisomy type of UPD by genome-wide single nucleotide polymorphism array. MYF5 was significantly downregulated by 69.14% (**p < 0.01) in HeLa cells transfected with mutant MYF5 containing c.191delC compared to those transfected with the wild-type MYF5, resulting in a truncated protein with a size of ∼20 kDa. In conclusion, this study identified a novel homozygous mutation in MYF5, broadening the genetic spectrum of EORVA and further deepening the understanding of this rare disease.

Novel Partial Exon 51 Deletion in the Duchenne Muscular Dystrophy Gene Identified via Whole Exome Sequencing and Long-Read Whole-Genome Sequencing.

Duchenne muscular dystrophy (DMD), one of the most common progressive and severely disabling neuromuscular diseases in children, can be largely attributed to the loss of function of the DMD gene on chromosome Xp21.2-p21.1. This paper describes the case of a 10-year-old boy diagnosed with DMD. Whole exome sequencing confirmed the hypothesized large partial exonic deletion of c.7310-11543_7359del (chrX:g.31792260_31803852del) spanning exon 51 and intron 50 in DMD. This large deletion was verified to be de novo by PCR, and the two breakpoints were further confirmed by Sanger sequencing and long-read whole-genome sequencing. Notably, this partial exonic deletion was the only complex variation in the deep intron regions or intron-exon junction regions in DMD. In addition, the case study demonstrates the clinical importance of using multiple molecular genetic testing methods for the diagnosis of rare diseases.