Structural Conservation Research Articles

Column layout design for concrete frame structures utilizing the strain energy-based topology optimization method

ABSTRACT Intelligent design is advancing civil engineering, providing new opportunities for structural optimization.This paper proposes an innovative intelligent design method integrating topology optimization into concrete frame column layout design.The method iteratively removes a defined percentage of columns from the initial layout, guided by the architectural plan, to achieve optimal column topologies.The approach’s efficacy is validated through a numerical example and a practical case study, showing that materials in optimized frames enhance structural stiffness more effectively than in traditional designs. Structures optimized via topology optimization can reduce material usage by 16% to 18% while preserving material properties.Moreover, comparing optimized designs at varying target volume fractions shows that initial fractions between 0.6 and 0.8 yield structures with robust performance and adequate stiffness.Thus, optimized column layouts allow structures to achieve maximum stiffness with fixed material or minimal material for specific stiffness requirements. Furthermore, the automated process enhances design efficiency and logic, reducing reliance on designer subjectivity. This method improves structural design efficiency, performance, and material conservation.

Shape Memory Alloys for Reversible Restoration of Ancient Monuments

Abstract The preservation of ancient structures through anastylosis requires methods that are both effective and reversible, while also preserving architectural integrity. Recent research into shape memory alloys (SMAs) offers a promising solution for reversible restoration, thanks to their ability to return to a predefined shape when exposed to thermal or mechanical triggers. However, challenges, such as cost, feasibility, and repeatability persist. This study focuses on characterizing the thermomechanical behavior of Nitinol SMAs under various conditions, with the aim of developing functional SMA devices for restoring monumental elements at the Valle dei Templi archeological site in Agrigento, Italy. The prototype’s superelastic properties and shape memory effects will be assessed to determine its effectiveness and applicability in different operational scenarios. This evaluation is grounded in a comprehensive analysis of the site's environmental conditions and an assessment of the alloy’s thermomechanical properties. By integrating SMAs into restoration efforts, the research aims to establish a methodology for creating reversible, replicable solutions that adhere to the principles of structural conservation.

The C2 domain augments Ras GTPase-activating protein catalytic activity.

Regulation of Ras GTPases by GTPase-activating proteins (GAPs) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity toward Ras. Analyses of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of GAPs for Ras.

Fine-scale population structure and widespread conservation of genetic effect sizes between human groups across traits.

Understanding genetic differences between populations is essential for avoiding confounding in genome-wide association studies and improving polygenic score (PGS) portability. We developed a statistical pipeline to infer fine-scale Ancestry Components and applied it to UK Biobank data. Ancestry Components identify population structure not captured by widely used principal components, improving stratification correction for geographically correlated traits. To estimate the similarity of genetic effect sizes between groups, we developed ANCHOR, which estimates changes in the predictive power of an existing PGS in distinct local ancestry segments. ANCHOR infers highly similar (estimated correlation 0.98 ± 0.07) effect sizes between UK Biobank participants of African and European ancestry for 47 of 53 quantitative phenotypes, suggesting that gene-environment and gene-gene interactions do not play major roles in poor cross-ancestry PGS transferability for these traits in the United Kingdom, and providing optimism that shared causal mutations operate similarly in different populations.

Comparative and phylogenetic analyses of the chloroplast genomes of mangrove plants

Mangrove ecosystems play a crucial role in coastal environment by stabilizing shorelines, reducing erosion, and providing habitat for diverse wildlife. However, there are fewer studies on the genetic management of mangrove plants. In this study, the chloroplast genome of Hibiscus tiliaceus L. was obtained through whole genome resequencing. The complete chloroplast genome of this species was 161,764 bp in length and comprised 84 protein-coding genes, eight rRNA (ribosomal RNA) genes, 37 tRNA (transfer RNA) genes. We conducted a comparative analysis of the chloroplast genomes of 26 mangrove species and found the conservation of genome structure among these mangrove plants, including the number of protein-coding genes, rRNA genes, and tRNA genes. However, there were noticeable differences in the number of SSRs (simple sequence repeats), the number of repeat sequences, and the borders of the IR (inverted repeat) regions, indicating genetic differentiation among mangrove plants. Besides, we found special differences in the distribution of certain genes in chloroplast genomes of certain species. Moreover, phylogenetic analysis based on chloroplast genome data clearly revealed that neither true mangrove plants nor semi-mangrove plants were monophyletic. These results revealed that chloroplast genomes will be useful to help understand the genetic differentiation and phylogenetic relationships of mangrove plants, which could assist in the genetic management, conservation, and restoration of mangrove ecosystems.

Structural conservation and functional role of TfpY-like proteins in type IV pilus assembly.

Type IV pili are surface filaments that enable versatile pathogens, like Pseudomonas aeruginosa, to adhere to and colonize surfaces. Pili are composed of diverse proteins called pilins, which serve as host receptors for phages. P. aeruginosa uses specific accessory proteins to glycosylate pilins to evade phage infection. Here, we show that TfpY is a conserved accessory protein that does not mediate phage defence. Instead, we propose a mechanism where TfpY facilitates efficient pilus assembly and function. A better understanding of TfpY function will provide insight into how its associated pilins have evolved to resist phage infection in the absence of post-translational modification, how some phages overcome this barrier to infection, and how this can guide the design of phage-based therapeutics.

Determination of the Negative Allosteric Binding Site of Cannabidiol at the CB1 Receptor: A Combined Computational and Site-Directed Mutagenesis Study.

Cannabinoid receptor 1 (CB1R) has been extensively studied as a potential therapeutic target for various conditions, including pain management, obesity, emesis, and metabolic syndrome. Unlike orthosteric agonists such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) has been identified as a negative allosteric modulator (NAM) of CB1R, among its other pharmacological targets. Previous computational and structural studies have proposed various binding sites for CB1R NAMs. An X-ray crystal structure revealed a binding site for the NAM, ORG27569, at an extrahelical location within the inner leaflet of the membrane. In contrast, multiple computational studies have previously proposed several potential allosteric binding sites for CBD within the CB1R structure. Given that a prior structural study suggested CBD might occupy the same site as ORG27569, we conducted a comprehensive investigation of potential CBD binding sites using molecular docking, molecular dynamics (MD) simulations, metadynamics (MTD) simulations, binding free-energy calculations, and in vitro mutagenesis experiments. Molecular docking, MD, and MTD simulations results, along with binding free-energy calculations, suggest that CBD may potentially bind to either the same extrahelical site as ORG27569 or a previously unidentified intracellular site located near TMHs 2, 6, and 7 and helix 8. This intracellular site is consistent with allosteric binding sites observed in other G protein-coupled receptors (GPCRs). To establish the most favorable allosteric site for CBD, we conducted site-directed mutagenesis of key residues at each site. Mutations at S4018.47ΔA and D4038.49ΔA augmented the binding of [3H]-SR141716A, suggesting these residues play critical roles in CBD binding. As a result, the combined computational and mutagenesis results identified a binding site for CBD between TMHs 2, 6, and 7 and helix 8, involving residues Y1532.40, I1562.43, M3376.29, L3416.33, S4018.47, and D4038.49. These findings provide valuable insights into how CBD binds to CB1R, thereby informing the rational design of new, selective, and potent NAMs. Moreover, the elucidation of this previously unexplored allosteric site might explain the polypharmacology of CBD due to structural conservation among Class A GPCRs.

Analysis of FBN1 gene mutations in six Chinese pedigrees affected with Marfan syndrome

To determine the types of genetic variants in six Chinese pedigrees affected with Marfan syndrome (MFS) and analyze their clinical characteristics and molecular pathogenesis. Six MFS pedigrees presented at the Taizhou Enze Medical Center (Group) between 2017 and 2022 were selected as the study subjects. Clinical data of pedigrees were retrospectively analyzed. Peripheral blood samples were collected from the probands and their family members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out. Candidate variants of the FBN1 gene were verified by Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), pathogenicity of the candidate variants was assessed. AlphaFold3 and PyMOL software were used for homology modeling of the FBN1 protein and analysis of its three-dimensional structure and amino acid sequence conservation. This study was approved by the Medical Ethics Committee of Taizhou Enze Medical Center (Group) (Ethics No. 20231002). Cardiovascular system abnormalities were noted in all pedigrees, ocular abnormalities were present in pedigrees 2 and 5, skeletal system abnormalities were presented in pedigrees 1, and 4 to 6. FBN1 gene mutations were identified in all pedigrees, including c.1957_1958dupGT (p.Asp654fs), c.5014T>A (p.Cys1672Ser), c.8135delC (p.Pro2712fs), c.2302G>T (p.Glu768*), c.3473A>G (p.Glu1158Gly) and c.6169C>T (p.Arg2057*), with each involving a different exon. Four variants were rated as pathogenic, one as likely pathogenic, and one as variant of uncertain significance. Among these, c.5014T>A (p.Cys1672Ser), c.1957_1958dupGT (p.Asp654fs), c.8135delC (p.Pro2712fs), and c.2302G>T (p.Glu768*) were unreported previously. Bioinformatic analysis with SIFT and PolyPhen-2 predicted that the c.5014T>A (p.Cys1672Ser) and c.3473A>G (p.Glu1158Gly) variants were deleterious. Protein homologous sequence alignment analysis revealed that the four novel mutation sites are highly conserved across various species. Homology modeling of the FBN1 protein three-dimensional structure indicated that the six variant sites in the amino acid sequence are all close to hydrogen bonds and may alter the secondary and tertiary structures to varying degrees, thereby confirmed the relationship between the variants and MFS. Four novel variants of the FBN1 gene have been discovered in this study, which has enriched the mutational and phenotypic spectrum of MFS and provided a basis for disease diagnosis and genetic counseling.

Comparative glucocorticoid receptor agonism: In silico, in vitro, and in vivo and identification of potential biomarkers for synthetic glucocorticoid exposure

Abstract The glucocorticoid receptor (GR) is present in almost every vertebrate cell and is utilized in many biological processes. Despite an abundance of mammalian data, the structural conservation of the receptor and cross-species susceptibility, particularly for aquatic species, has not been well defined. Efforts to reduce, refine, and/or replace animal testing have increased, driving the impetus to advance development of new approach methodologies (NAMs). Here we used in silico, in vitro, and in vivo methods to elucidate a greater understanding of receptor-mediated effects of synthetic glucocorticoid exposure in teleost fish. Evolutionary conservation of amino acid residues critical for transcriptional activation was confirmed in silico using sequence alignment to predict across species susceptibility. Subsequent in vitro assays using zebrafish and human GR provided evidence of physiological congruence of GR agonism. Finally, adult fathead minnows (Pimephales promelas) were exposed in vivo to the synthetic glucocorticoids, dexamethasone (0.04, 400, 4,000 µg/L) and beclomethasone dipropionate (130 µg/L), and GR agonism confirmed via digital polymerase chain reaction; in addition, EcoToxChip analyses identified potential mRNA biomarkers following glucocorticoid exposure. These findings support the use of NAMs to potentially reduce multispecies in vivo experimentation while providing empirical evidence that expands the taxonomic domain of applicability for the GR agonism molecular initiating event within the broader GR agonism adverse outcome pathway network.

Conservation of structure and dynamic behavior in triazine macrocycles with opportunities for subtle control of hinge motion.

Modifying the backbone of 24-atom macrocycles allows tailoring of physical properties (octanol-water partition coefficients, log P) while conserving both conformation and the barrier to dynamic, hinge-like motion. Structure is determined by 1D and 2D NMR spectroscopy. The barrier can be subtly tuned by modifications that appear to preclude fully revolute motion and efficient π-stacking of the two subunits. The log P values increase as expected across this series, and the correlation between computed and observed values reinforces the belief of common structure and behavior across this class of macrocycles.

In silico protein structural analysis of PRMT5 and RUVBL1 mutations arising in human cancers.

DNA double strand breaks (DSBs) can be generated spontaneously during DNA replication and are repaired primarily by Homologous Recombination (HR). However, efficient repair requires chromatin remodeling to allow the recombination machinery access to the break. TIP60 is a complex conserved from yeast to humans that is required for histone acetylation and modulation of HR activity at DSBs. Two enzymatic activities within the TIP60 complex, KAT5 (a histone acetyltransferase) and RUVBL1 (an AAA+ ATPase) are required for efficient HR repair. Post-translational modification of RUVBL1 by the PRMT5 methyltransferase activates the complex acetyltransferase activity and facilitates error free HR repair. In S. pombe a direct interaction between PRMT5 and the acetyltransferase subunit of the TIP60 complex (KAT5) was also identified. The TIP60 complex has been partially solved experimentally in both humans and S. cerevisiae, but not S. pombe. Here, we used in silico protein structure analysis to investigate structural conservation between S. pombe and human PRMT5 and RUVBL1. We found that there is more similarity in structure conservation between S. pombe and human proteins than between S. cerevisiae and human. Next, we queried the COSMIC database to analyze how mutations occurring in human cancers affect the structure and function of these proteins. Artificial intelligence algorithms that predict how likely mutations are to promote cellular transformation and immortalization show that RUVBL1 mutations should have a more drastic effect than PRMT5. Indeed, in silico protein structural analysis shows that PRMT5 mutations are less likely to destabilize enzyme function. Conversely, most RUVBL1 mutations occur in a region required for interaction with its partner (RUVBL2). These data suggests that cancer mutations could destabilize the TIP60 complex. Sequence conservation analysis between S. pombe and humans shows that the residues identified in cancer cells are highly conserved, suggesting that this may be an essential process in eukaryotic DSB repair. These results shed light on mechanisms of DSB repair and also highlight how S. pombe remains a great model system for analyzing DSB repair processes that are tractable in human cells.

Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons.

Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110-family transposases, which catalyse RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-O-methylation using C/D-box snoRNAs. On the basis of conservation of protein sequence, domain architecture, three-dimensional structure and non-coding RNA features, alongside phylogenetic analyses, we propose that programmable RNA modification emerged through the exaptation of components derived from IS110-like transposons. These findings underscore how recurrent domestication events of transposable elements have driven the evolution of RNA-guided mechanisms.

Dysregulated Long Non-coding RNAs in Myasthenia Gravis- A Mini-Review.

Myasthenia gravis (MG) is an acquired autoimmune disease that is mediated by humoral immunity, supplemented by cellular immunity, along with participation of the complement system. The pathogenesis of MG is complex; although autoimmune dysfunction is clearly implicated, the specific mechanism remains unclear. Long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecules with lengths greater than 200 nucleotides, with increasing evidence of their rich biological functions and high-level structure conservation. LncRNAs can directly interact with proteins and microRNAs to regulate the expression of target genes at the transcription and post-transcription levels. In recent years, emerging studies have suggested that lncRNAs play roles in the differentiation of immune cells, secretion of immune factors, and complement production in the human body. This suggests the involvement of lncRNAs in the occurrence and progression of MG through various mechanisms. In addition, the differentially expressed lncRNAs in peripheral biofluid may be used as a biomarker to diagnose MG and evaluate its prognosis. Moreover, with the development of lncRNA expression regulation technology, it is possible to regulate the differentiation of immune cells and influence the immune response by regulating the expression of lncRNAs, which will provide a potential therapeutic option for MG. Here, we review the research progress on the role of lncRNAs in different pathophysiological events contributing to MG, focusing on specific lncRNAs that may largely contribute to the pathophysiology of MG, which could be used as potential diagnostic biomarkers and therapeutic targets.

Placement of dental implants using 3D surgical guide

Background. The procedures of dental implant application can lead to optimal clinical outcomes, being simplified by the guided implantology protocol. In the latest articles from specialized literature it is said that digital planning of implants ensures the most accurate diagnosis of the area where the implant will be positioned, with clinical benefits including reduced operative time and a lower rate of complications, all of which can ultimately lead to better acceptance and greater patient satisfaction. Case report. A 55-years-old female patient came to the clinic for a full mandibula rehabilitation. In this situation, the patient wanted a solution using only fixed restoration so the treatment plan that the patient agreed to was an all-on-X restoration. Aim. Given the constant evolution of technology, along with the development of dental technical laboratories, this paper wants to prove that the latest technology can help doctors in placing the dental implants accurately and in a more comfortable manner for both the patient and the doctor. Material and methods. For this case, the insertion of dental implants was performed using the static approach, which refers to the use of tissue-supported surgical guides; this replicates the virtual position of the implant directly from the data provided by Cone Beam Computed Tomography (CBCT). This information was converted into a surgical guide that was used during the surgical procedure. Before the surgical intervention, a CBCT scan was performed, which was used along with the patient's intraoral scanning to establish the ideal position of each dental implant. After the surgical intervention and after the complete healing, another CBCT scan was conducted. Thus, to analyze the accuracy of dental implant insertion, radiological investigations were compared to see the differences between the ideal situation planned before the surgery and the situation resulting from the surgery. Results. The results demonstrate a high degree of accuracy in the insertion of dental implants using the three-dimensional surgical guide, without significant positioning differences. Conclusions. These outcomes support the main purpose of three-dimensional surgical guides, namely the conservation of anatomical structures at the level of the dento-maxillary system.

IN SILICO ANALYSIS OF IDENTIFIED MOST FREQUENT MUTATIONS IN EXON 10, CODON 618 OF THE HUMAN RET GENE

Background: The Rearranged during Transfection (RET) gene mutations are known to be responsible for Hirschsprung disease (HSCR), as well as the multiple endocrine neoplasia (MEN) types 2A, 2B and familial medullary thyroid carcinoma (FMTC). Aim: In this study we analyse mutations of codon 618 (Cys618Phe, Cys618Arg, Cys618Ser, Cys618Tyr) in exon 10, in terms of their structural similarities, stability, conservation and interaction with their close interactor GFRA3 protein. Material and Methods: In the present study, multiple bioinformatics software was used, including HGMD and UniProt for the data collection. The structural prediction of different mutations at codon 618 was performed by using PSIPRED for secondary structure prediction and I-TASSER for 3D structure modelling. The mutated proteins stability was determined, using I- Mutant and MuPro, while the evolutionary amino acids conservation was assessed with the ConSurf tool. The wildtype RET and mutant proteins were independently docked with their nearby interactor protein GFRA3.Findings: The analysis indicates that among the examined mutations, the Cys618Phe displayed the greatest structural and functional similarity to the wildtype RET protein. The interaction analysis suggested that the Cys618Phe demonstrated stronger binding with GFRA3 compared to other mutations. Conclusion: This study highlights the impact of codon 618 mutations in exon 10 of the RET gene on protein stability, structure, and interactions with GFRA3. We find mutation Cys618Phe comparatively similar to RET wildtype protein while dissimilar mutation Cys618Tyr. These findings could help a molecular geneticist in analyzing mutations at codon 618 for both research and diagnostic purpose.

Genome-Wide Identification and Characterization of GRAS Transcription Factor Family in Cultivated Hybrid Sugarcane ZZ1 (Saccharum officinarum) and Their Role in Development and Stress.

GRAS gene family plays multifunctional roles in plant growth, development, and resistance to various biotic and abiotic stresses, belonging to the plant-specific transcription factor (TF) family. In this study, a genome-wide survey and systematic analysis of the GRAS family in cultivated hybrid sugarcane ZZ1 (Saccharum officinarum) with economic and industrial importance was carried out. We identified 747 GRAS genes with complete structural domains and classified these into 11 subfamilies by phylogenetic analyses, exhibiting a diverse range of molecular weight and isoelectric points, thereby indicating a broad structural and functional spectrum. Analysis of Protein motif and gene structure revealed a conserved yet variable arrangement of motifs within the GRAS TFs, suggesting its potential for diverse functional roles. Furthermore, the identification of numerous cis-regulatory elements by GRAS TFs promoter sequence analysis, implying their complex regulation in response to environmental and physiological signals. Tertiary structure predictions analyses using AlphaFold3 highlighted the structural flexibility and conservation within the GRAS family, with disordered regions potentially contributing to their functional versatility. Weighted Gene Co-expression Network Analysis (WGCNA) provided insights into the potential roles of ShGRAS21A in sugarcane's response to smut infection. This comprehensive investigation of the GRAS family in ZZ1 not only uncovers their structural diversity but also sheds light on their potential regulatory roles in plant growth, development, and stress response. The findings contribute to a deeper understanding of GRAS TFs functions and lay the groundwork for future studies on their role in sugarcane improvement and disease resistance.

Targeted protein degradation of PDE4 shortforms by a novel proteolysis targeting chimera.

Cyclic AMP (cAMP) has a crucial role in many vital cellular processes and there has been much effort expended in the discovery of inhibitors against the enzyme superfamily that degrades this second messenger, namely phosphodiesterases (PDEs). The journey of competitive PDE inhibitors to the clinic has been hampered by side effects profiles that have resulted from a lack of selectivity for subfamilies and individual isoforms because of high conservation of catalytic site sequences and structures. Here we introduce a proteolysis targeting chimera (PROTAC) that can specifically target a small subset of isoforms from the PDE4 family to send the enzyme for degradation at the proteasome by recruiting a ubiquitin E3 ligase into proximity with the PDE. We constructed our PDE4 PROTAC (KTX207) using a previously characterized PDE4 inhibitor, and we show that evolution of the compound into a PROTAC improves selectivity, potency and enables a long-lasting effect even after the compound is removed from cells after a short treatment duration. Functionally, KTX207 is more effective at increasing cAMP, is 100 times more anti-inflammatory, and is significantly better at reducing the growth in cancer cell models than the PDE4 inhibitor alone. Our study highlights the advantages of targeted degradation versus active-site occupancy for PDE4 inhibition and discusses the potential of this novel pharmacological approach to improve the safety profile of PDE4 inhibition in the future.

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4‐Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites

AbstractCyanotryptophans (CN‐Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl‐tRNA synthetases specific for 4‐, 5‐, 6‐, and 7‐CN‐Trp for high‐yield in vivo production of proteins with a single, site‐specifically introduced nitrile label. The absorption maximum of 4‐CN‐Trp is distinct from Trp, allowing the selective excitation of its intense fluorescence. 4‐CN‐Trp fluoresces in the visible range with an intensity rivalling that of 7‐hydroxy‐coumarin. Crystal structures of maltose binding protein demonstrate near‐complete structural conservation when a native buried Trp residue is replaced by 4‐CN‐Trp. Besides presenting an inconspicuous tag for live cell microscopy, the intense fluorescence of 4‐CN‐Trp enables measurements of subnanomolar ligand binding affinities in isotropic solution, as demonstrated by the complex between rapamycin and the peptidyl–prolyl isomerase FKBP12 furnished with a 4‐CN‐Trp residue in the substrate binding pocket. Furthermore, 4‐CN‐Trp residues positioned at different locations of a protein containing multiple tryptophan residues permits using fluorescence quenching experiments to detect the proximity of individual Trp residues to the binding site of aromatic ligands.

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4-Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites.

Cyanotryptophans (CN-Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl-tRNA synthetases specific for 4-, 5-, 6-, and 7-CN-Trp for high-yield in vivo production of proteins with a single, site-specifically introduced nitrile label. The absorption maximum of 4-CN-Trp is distinct from Trp, allowing the selective excitation of its intense fluorescence. 4-CN-Trp fluoresces in the visible range with an intensity rivalling that of 7-hydroxy-coumarin. Crystal structures of maltose binding protein demonstrate near-complete structural conservation when a native buried Trp residue is replaced by 4-CN-Trp. Besides presenting an inconspicuous tag for live cell microscopy, the intense fluorescence of 4-CN-Trp enables measurements of subnanomolar ligand binding affinities in isotropic solution, as demonstrated by the complex between rapamycin and the peptidyl-prolyl isomerase FKBP12 furnished with a 4-CN-Trp residue in the substrate binding pocket. Furthermore, 4-CN-Trp residues positioned at different locations of a protein containing multiple tryptophan residues permits using fluorescence quenching experiments to detect the proximity of individual Trp residues to the binding site of aromatic ligands.

Comparative Genomic Analysis of Bacillus velezensis BRI3 Reveals Genes Potentially Associated with Efficient Antagonism of Sclerotinia sclerotiorum (Lib.) de Bary.

Bacillus velezensis has recently received increased attention as a potential biological agent because of its broad-spectrum antagonistic capacity against harmful bacteria and fungi. This study aims to thoroughly analyze the genomic characteristics of B. velezensis BRI3, thereby providing theoretical groundwork for the agronomic utilization of this strain. In this work, we evaluated the beneficial traits of the newly isolated strain B. velezensis BRI3 via in vitro experiments, whole-genome sequencing, functional annotation, and comparative genomic analysis. B. velezensis BRI3 exhibits broad-spectrum antifungal activity against various soilborne pathogens, displays inhibitory effects comparable to those of the type strain FZB42, and exhibits particularly effective antagonism against Sclerotinia sclerotiorum (Lib.) de Bary. Whole-genome sequencing and assembly revealed that the genome of BRI3 contains one chromosome and two plasmids, which carry a large amount of genetic information. Moreover, 13 biosynthetic gene clusters (BGCs) involved in the biosynthesis of secondary metabolites were predicted within the BRI3 genome. Among these, two unique BGCs (cluster 11 and cluster 13), which were not previously reported in the genomes of other strains and could potentially encode novel metabolic products, were identified. The results of the comparative genomic analysis demonstrated the genomic structural conservation and genetic homogeneity of BRI3. The unique characteristics and genomic data provide insights into the potential application of BRI3 as a biocontrol and probiotic agent.