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Substrate recognition by the 4-hydroxytryptamine kinase PsiK in psilocybin biosynthesis.

Psilocybin, the natural hallucinogen from Psilocybe (magic) mushrooms, is a highly promising drug candidate for the treatment of depression and several other mental health conditions. Biosynthesis of psilocybin from the amino acid l-tryptophan involves four strictly sequential modifications. The third of these, ATP-dependent phosphorylation of the intermediate 4-hydroxytryptamine, is catalysed by PsiK. Here we present a crystallographic analysis and a structure-based mutagenesis study of this kinase, providing insight into its mode of substrate recognition. The results of our work will support…

Ribbon Synapses and Retinal Disease: Review.

Synaptic ribbons are presynaptic protein complexes that are believed to be important for the transmission of sensory information in the visual system. Ribbons are selectively associated with those synapses where graded changes in membrane potential drive continuous neurotransmitter release. Defective synaptic transmission can arise as a result of the mutagenesis of a single ribbon component. Visual diseases that stem from malfunctions in the presynaptic molecular machinery of ribbon synapses in the retina are rare. In this review, we provide an overview of synaptopathies that give rise to reti…

Nonintegrating Gene Therapy Vectors.

Gene delivery vectors that do not rely on host cell genome integration offer several advantages for gene transfer, chiefly the avoidance of insertional mutagenesis and position effect variegation. However, unless engineered for replication and segregation, nonintegrating vectors will dilute progressively in proliferating cells, and are not exempt of epigenetic effects. This article provides an overview of the main nonintegrating viral (adenoviral, adeno-associated viral, integration-deficient retro-lentiviral, poxviral), and nonviral (plasmid vectors, artificial chromosomes) vectors used for p…

The impact of quasispecies dynamics on the use of therapeutics.

The application of quasispecies theory to viral populations has boosted our understanding of how endogenous and exogenous features condition their adaptation. Mounting empirical evidence demonstrates that internal interactions within mutant spectra may cause unexpected responses to antiviral treatments. In this scenario, increased mutagenesis could be efficient at low mutagen doses due to the lethal action of defective genomes, whereas sequential administration of antiviral drugs might be superior to combination therapies. Our ability to predict the outcome of a particular therapy takes advant…

Kidney development and disease in the zebrafish.

Unraveling the molecular pathogenesis of human disease presents many experimental challenges, not the least of which is that experiments on humans are generally frowned upon. Model organisms, including the zebrafish, allow for experimental analysis of gene function and the detailed characterization of disease processes. Zebrafish have matured as a vertebrate model organism now that genetic tools for targeted "knockdowns" and unbiased mutagenesis approaches are in hand. The fish larval pronephros is a relevant kidney in which to pursue many aspects of human kidney development and disease. This …

Subunit-specific block of cloned NMDA receptors by argiotoxin636.

Cloned NMDA receptor channels of the NR1-NR2A, NR1-NR2B and NR1-NR2C type show differences in argiotoxin636 block. Mutations of an asparagin … not responsible for the subtype-specific sensitivity to argiotoxin636 as revealed by site-directed mutagenesis.

APC gene messenger RNA: novel isoforms that lack exon 7.

The APC gene at human chromosome 5q21 is responsible for familial adenomatous polyposis coli. Furthermore, sporadic cancers of not only colon but also other digestive organs often contain mutations in the APC gene. A dominant mouse mutation Min that was generated by chemical mutagenesis and causes polyposis in the digestive tract is in the mouse homologue of the human APC gene. The APC mRNA is generated from 15 exons. Two mRNA isoforms were reported which are produced by alternative splicing in the 9th exon. Here, we report novel mRNA isoforms that lack the 7th exon in both mouse and human cel…

mTREX2 co-expression promotes large deletions in CRISPR/Cas12a editing of the highly polyploid sugarcane genome.

The LbCas12ai-mTREX2 system enhances large deletions, and the LbCas12ai-RRV enables efficient editing of lw1 in sugarcane. Sugarcane (Saccha … rgeting AT-rich genomic regions. In this study, we report efficient CRISPR/Cas12a-mediated targeted mutagenesis of the lw1 gene in sugarcane. The lemon white1 (lw1) is a key regulator of chlorophyll and carotenoid biosynthesis, and targeted lw1 mutagenesis results in a visible phenotype. We compared the editing performance of intron-containing LbCas12a-R (LbC …

Optimized flow cytometry assay for functional characterization of variants of uncertain significance in familial hypercholesterolemia.

BACKGROUND: The definitive diagnosis of Familial Hypercholesterolemia (FH) relies on the identification of deleterious variants in FH-relate … type in at least one parameter. For a VUS subset, LDLR activity was further tested by site-directed mutagenesis and optical microscopy. RESULTS: At flow cytometry assay, 15 variants (75%) showed deleterious effects in at least one functional LDLR parameter; the others affected expression (n = 5; 35.7%) or binding/expression or binding/uptake (n = 6; 42.8%); …

CRISPR/Cas9 ribonucleoprotein-mediated knockout of Gly m 4-L1 eliminates allergen accumulation in soybean.

Gly m 4 is a major allergen in soy milk and cross-reacts with Bet v 1, the primary birch pollen allergen. To reduce Gly m 4 allergenicity, we performed site-directed mutagenesis of the Gly m 4 gene in soybean through a DNA-free in planta bombardment method using ribonucleoproteins (iPB-RNP). Although a mutant line (Gly m 4-2del ) was generated by targeting the Gly m 4-2 gene, immunoblot analysis revealed that translation products from other homologs still accumulated in the seed tissues. Subsequent gene expression analysis identified Gly m 4-L1, one of eight Gly m 4 homologs in the soybean gen…

GII.23/24/25 noroviruses recognize glycans via a conventional glycan-binding site.

INTRODUCTION: Human noroviruses (HuNoVs) are genetically diverse RNA viruses that cause acute gastroenteritis, with genogroup II (GII) accou … of their P domains, including the GII.25 P domain in complex with the H disaccharide. Single-point mutagenesis was performed to identify key residues involved in glycan binding. RESULTS: The P domains of GII.24 and GII.25 can recognize multiple types of saliva samples, including both A/B/O secretor and nonsecretor individuals. In contrast, GII.23 primarily …

Enhancing agronomic traits and bioactive compound accumulation in Thai Andrographis paniculata via EMS-induced variation.

BACKGROUND: Andrographis paniculata is a self-pollinating medicinal plant with limited natural genetic diversity, which restricts the development of improved cultivars with desirable agronomic traits and stable accumulation of key bioactive compounds, particularly andrographolide (AP1). To overcome this limitation, ethyl methanesulfonate (EMS) mutagenesis was applied to native Thai germplasm to induce phenotypic and phytochemical variation and to generate novel genetic resources for breeding improvement and gene function analysis. RESULTS: Optimal EMS treatment conditions (1.0% and 1.5% EMS fo…

Defining AV2-1 as a novel pharmacological probe to target human and rodent TRPV2.

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid 2 (TRPV2) is a non-selective cation channel implicated in immune cell functio … gical recordings. Structural insights were obtained from cryoEM of TRPV2 in complex with AV2-1, and mutagenesis was performed to confirm binding site residues. The efficacy of AV2-1 was assessed in human peripheral blood-derived macrophages by Ca2+ imaging, whole-cell electrophysiology and TIRF microscopy to detect Ca2+ microdomains. KEY RESULTS: AV2-1 is a …

Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers.

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and β chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies deta…

Characteristics of Helicase-Primase Inhibitor Amenamevir-Resistant Herpes Simplex Virus.

The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster … V-resistant HSV-1 with a single amino acid substitution using bacterial artificial chromosome (BAC) mutagenesis. As a result, G352C in UL5 helicase and F360C/V and N902T in UL52 primase were identified as novel mutations. The virus with K356N in UL5 showed 10-fold higher AMNV resistance than did other mutants and showed equivalent viral growth in vitro and vi …

Antibiotic susceptibility of clinical Burkholderia pseudomallei isolates in northeast Thailand during 2015-2018 and the genomic characterization of β-lactam-resistant isolates.

Melioidosis is an often fatal infection in tropical regions caused by an environmental bacterium, Burkholderia pseudomallei Current recommen … e analysis, penA expression and β-lactamase activity. The resistant phenotype was verified via penA mutagenesis. All primary isolates were IPM-susceptible but we observed two CAZ-resistant and one CAZ-intermediate resistant isolates, two MEM-less susceptible isolates, one AMC-resistant and two AMC-intermediate resistant isolates. One of 13 relapse isolates wa …

Effect of mutation on structure, function and dynamics of receptor binding domain of human SARS-CoV-2 with host cell receptor ACE2: a molecular dynamics simulations study.

Recent studies have pointed the role of angiotensin-converting enzyme-II (ACE2) in mediating the entry of SARS-CoV-2 to the host cell by bin … tor binding motif (RBM) of the ACE2-RBD SARS-CoV-2 complex (PDB: 6M0J). Computational site directed mutagenesis induced dynamics in wild-type and mutant complexes were extensively studied through all-atoms molecular dynamics (MD) simulations of 150 ns. In silico mutational analysis revealed loss of important intermolecular hydrogen bonds and other non-bonded …

Перенос и окончательное высвобождение мини-Ф вектора клонированного инфекционного герпесвирусного генома.

Бактериальные искусственные хромосомы (КБК) являются хорошо зарекомендовавшими себя средствами клонирования для функциональной геномики и для построения целевых векторов и инфекционных вирусных клонов ДНК. Методы мутагенеза, основанные на рекомбинации красного цвета, позволили манипулировать BAC в Escherichia coli без каких-либо оставшихся операционных последовательностей. Здесь мы описываем, что векторные последовательности, полученные с помощью F-факторов, могут быть вставлены в новое положение и бесперебойно удалены из нынешнего местоположения клонированной ДНК BAC посредством синхронизации

Совпадение в аннотации оригинала
Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-…

The DNA polymerase gamma Y955C disease variant associated with PEO and parkinsonism mediates the incorporation and translesion synthesis opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Mitochondrial DNA is replicated and repaired by DNA polymerase gamma (pol gamma), encoded by the POLG gene. The Y955C substitution in POLG l … can react with dG to form 8-oxo-dG as one of the more common products. DNA polymerases can mitigate mutagenesis by 8-oxo-dG through allosteric interactions from amino acid side chains, which limit the anti-conformation of the 8-oxo-dG template base during translesion DNA synthesis. Here, we show that the Y955C pol gamma displays relaxed discrimination when ei …

ADAMTS13 substrate recognition of von Willebrand factor A2 domain.

ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in theA2 domain. To ex … teolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved approximately 8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614 on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K16 …