Tool For Modification Research Articles

Discrete Hybrid Vanadium-oxo Cluster as a Targeted Tool for Selective Protein Oxidative Modifications and Cleavage.

Understanding the impact of oxidative modification on protein structure and functions is essential for developing therapeutic strategies to combat macromolecular damage and cell death. However, selectively inducing oxidative modifications in proteinsremains challenging. Herein we demonstrate that [V6O13{(OCH2)3CCH2OH}2]2-(V6-OH) hybrid metal-oxo cluster can be used for selective protein oxidative cleavage and modifications. We present the first example of a protein-bound hybrid vanadate cluster, whereinteractions with protein surfaces and the redox activity of vanadium enable selective oxidative modifications. Single Crystal X-ray Diffraction (SC-XRD) of the V6-OH and hen egg white lysozyme (HEWL) complex revealed that the binding is dictated both by the inorganic core and the organic ligands attached to it. Selective modifications of HEWL occurs under physiological conditions by producing reactive oxygen species in presence of ascorbate as a reducing agent. The oxidative reaction can be tuned by varying the concentration of V6-OH to result either in selective oxidation of side chains or peptide bond cleavage. LC-MS and crystallography revealed that oxidative modifications were mainly concentrated near the cluster binding sites, providing spatial control of ROS production. This study advances our understanding of vanadium role in biological systems and demonstrates the potential of hybrid metal-oxo clusters in protein modification.

Discrete Hybrid Vanadium‐oxo Cluster as a Targeted Tool for Selective Protein Oxidative Modifications and Cleavage

Discrete Hybrid Vanadium‐oxo Cluster as a Targeted Tool for Selective Protein Oxidative Modifications and Cleavage

Myceliophthora thermophila as promising fungal cell factories for industrial bioproduction: From rational design to industrial applications.

Myceliophthora thermophila stands out as a prominent fungal cell factory, garnering growing interest due to its distinctive traits advantageous. Currently, M. thermophila has been developed as an efficient cell factory, producing a variety of products from various raw materials. In this review, we firstly discuss the potential advantages of M. thermophila as a platform for metabolic engineering and industrial applications, with special emphasis on its physiological characteristics, the development of genetic modification techniques and tools, gene expression and regulation strategies. Then, the latest progress in industrial application of M. thermophila as microbial cell factory was systematically summarized, including biochemical synthesis platform, enzyme expression platform, antibody protein and vaccine production platform, bio-organic fertilizer production platform, and efficient enzyme element library. Finally, the current challenges of M. thermophila as a cell factory and its corresponding strategies are proposed, aiming to achieve green biomanufacturing of multiple products with higher efficiency.

TSPO-PET Reveals Higher Inflammation in White Matter Disrupted by Paramagnetic Rim Lesions in Multiple Sclerosis.

To explore whether the inflammatory activity is higher in white matter (WM) tracts disrupted by paramagnetic rim lesions (PRLs) and if inflammation in PRL-disrupted WM tracts is associated with disability in people with multiple sclerosis (MS). Forty-four MS patients and 16 healthy controls were included. 18 kDa-translocator protein positron emission tomography (TSPO-PET) with the 11 C-PK11195 radioligand was used to measure the neuroinflammatory activity. The Network Modification Tool was used to identify WM tracts disrupted by PRLs and non-PRLs that were delineated on MRI. The Expanded Disability Status Scale was used to measure disability. MS patients had higher inflammatory activity in whole brain WM compared to healthy controls (p=0.001). Compared to patients without PRLs, patients with PRLs exhibited higher levels of inflammatory activity in the WM tracts disrupted by any type of lesions (p=0.02) or PRLs (p=0.004). In patients with at least one PRL, inflammatory activity was higher in WM tracts highly disrupted by PRLs compared to WM tracts highly disrupted by non-PRLs (p=0.009). Elevated inflammatory activity in highly disrupted WM tracts was associated with increased disability in patients with PRL (p=0.03), but not in patients without PRL (p=0.2). This study suggests that patients with PRLs may exhibit more diffuse WM inflammation in addition to higher inflammation along WM tracts disrupted by PRLs compared to non-PRLs, which could contribute to larger lesion volumes and faster disability progression. Imaging PRLs may serve to identify patients with both focal and diffuse inflammation, guiding therapeutic interventions aimed at reducing inflammation and preventing progressive disability in MS.

Trends in biotechnology: <em>Vibrio natriegens</em> as potential micro-factory for valorization of crustacean waste

Vibrio natriegens has recently been identified as a promising host for the biotechnology industry thanks to its inherent qualities, which include its fast growth rate, non-pathogenicity to humans, and versatility in using substrates. These advantages have led to the potential use of V. natriegens in the biosynthesis of several products. Basically, the industrial scale requires fermentation or cultivation processes to be conducted at high substrate or biomass concentrations to maximize the final retrieved product. However, studies on V. natriegens at high cell density are limited. Besides, the potential of V. natriegens to convert recalcitrant substrates such as chitin derivatives into biological products has not yet been understood. This review summarizes up-to-date information on the physiological characteristics, metabolism, genome, and genetic modification tools of V. natriegens. Subsequentially, statistics and analysis of research trends related to V. natriegens was presented. Finally, a discussion on the role of V. natriegens in converting chitin waste from the seafood processing industry into a culturing feedstock to achieve a circular economy and net zero emissions was provided.

The Role of Externalizing Problems and Empathy on the Daily Report Card

AbstractThe daily report card (DRC) is a commonly used behavioral intervention in which teachers rate child performance on target goals and parents provide home rewards based on the child’s performance. The current study investigated associations between child externalizing problems, empathy, and specific components of the DRC: (1) types of DRC goals that are chosen, (2) teacher and parent adherence to the DRC, and (3) child performance on the DRC. These aims were examined in a sample of 71 children (ages 7–11) who were enrolled in a school-home intervention designed for children with significant inattentive and hyperactive/impulsive behaviors. Teachers and parents completed separate baseline measures of inattention (IA), hyperactivity/impulsivity (H/I), conduct problems (CP), and empathy. Outcomes included type of DRC goals (i.e., academic, behavioral, and social-emotional), teacher and parent adherence to the DRC during the initial four-week intervention period, and child DRC performance during the same period. First, IA predicted more academic goals but fewer social-emotional goals, H/I predicted fewer academic goals but more behavioral goals, and CP predicted more social-emotional goals. Second, H/I predicted better parental adherence such that parents rewarded their child’s DRC more frequently. Third, baseline empathy predicted better overall DRC performance; externalizing problems did not negatively impact DRC performance. Results suggest that the DRC is a robust behavioral modification tool that can be tailored to fit each child’s needs and severity of externalizing problems. Empathy may serve as an important factor when designing treatment protocols to improve overall child behavior.

Targeted epigenome engineering

Cellular differentiation and homeostasis rely on complex mechanisms to control gene expression, enabling the different cell lineages of an organism to establish and then "memorize" different epigenetic states. The processes that control gene expression are centered on chromatin, a complex of DNA, histone proteins and RNA, whose structure is finely regulated. Targeted epigenomic engineering tools make it possible to interfere with and study these processes, revealing the logic of epigenetic memory mechanisms. This article reviews the main classes of targeted epigenome modification tools and illustrates how they can be used to better understand and modify the epigenome of cells, paving the way for potentially revolutionary therapeutic prospects.

An efficient and easily obtainable butelase variant for chemoenzymatic ligation and modification of peptides and proteins

The expanding field of site-specific ligation of proteins and peptides has catalyzed the development of novel methods that enhance molecular modification. Among these methods, enzymatic strategies have emerged as dominant due to their specificity and efficiency in modifying proteins under mild conditions. Asparaginyl endopeptidase is a group of cyclotide-producing cysteine proteases from plants. These plant cysteine proteases, known for their specificity, effectively recognize the tripeptide motif (Asx-Xaa-Yaa) and cleave at the C-terminal side of Asx residues, forming acyl-enzyme intermediates that facilitate transpeptidation. Butelase 1 stands out as the most efficient AEP for protein engineering, yet challenges in its expression and purification limit its accessibility for widespread research and industrial use. To address these challenges, we engineered a new, catalytically efficient variant of Butelase 1, Butelase AY, by mutating the gatekeeping residues Val237Ala and Thr238Tyr within the LAD-1 region. These modifications significantly enhanced the stability and yield of Butelase AY, allowing for successful application in various peptide and protein engineering tasks. Butelase AY was tested on the peptide GLGKY, the globular protein GFP, and the intrinsically disordered protein α-synuclein, effectively labeling them with a fluorescent probe. Notably, Butelase AY maintained its efficiency with substrates containing unnatural amino acids, making it a promising candidate for biorthogonal applications. Importantly, the mutations did not compromise the enzyme’s specificity, as it continued to process model peptides and native protein substrates with N-term NHV recognition motifs effectively. In conclusion, Butelase AY presents a novel recombinant tool for diverse protein labeling and modifications, particularly in biorthogonal strategies. This innovation has the potential to expand applications in biotechnology and therapeutic development, ultimately revolutionizing protein engineering and its utility in synthetic biology.

ZnO nanoparticles enhances cadmium tolerance by modulating N6-methyladenosine (m6A) level of stress-responsive genes NRT1 and GM35E in vegetable soybean

Cadmium (Cd) is a hazardous heavy metal pollutant that poses significant risks to agricultural production and human health. Nanoparticles (NPs) can alleviate the effects of cadmium on crops by regulating the expression of stress-responsive genes, however, the mechanism of regulation is unknown. N6-methyladenosine (m6A) is a prevalent RNA modification, which determines the expression level of RNA. In this study, we performed m6A methylome analysis and gene editing to investigate the regulation of stress-responsive genes by m6A under Cd stress with ZnO nanoparticles (ZnO NPs). Firstly, we identified 16 differentially expressed genes (DEGs) with differential m6A-modification by m6A methylome seq, which included 6 stress-responsive genes. ZnO NPs treatment reduced the m6A level and stabilized the mRNAs of these stress-responsive genes. Then, we utilized the in vivo m6A modification tool, Plant m6A Editors (PMEs), specifically reduced the m6A level of NRT1 and GM35E in transgenic plants. The expression of NRT1 and GM35E was increased, and plants carrying PMEs-NRT1 and PMEs-GM35E showed stronger Cd tolerance than control. Therefore, we can conclude that NPs enhance Cd tolerance in plants by regulating the m6A methylation level of stress-responsive genes such as NRT1 and GM35E, which ultimately affects the expression of these genes. This study proposed a post-transcriptional regulatory network in vegetable soybean roots responding to Cd with ZnO NPs, potentially offering new insights into gene manipulation for controlling low Cd accumulation in crops.

Landscape burning facilitated Aboriginal migration into Lutruwita/Tasmania 41,600 years ago.

The establishment of Tasmanian Palawa/Pakana communities ~40 thousand years ago (ka) was achieved by the earliest and farthest human migrations from Africa and necessitated migration into high-latitude Southern Hemisphere environments. The scarcity of high-resolution paleoecological records during this period, however, limits our understanding of the environmental effects of this pivotal event, particularly the importance of using fire as a tool for habitat modification. We use two paleoecological records from the Bass Strait islands to identify the initiation of anthropogenic landscape transformation associated with ancestral Palawa/Pakana land use. People were living on the Tasmanian/Lutruwitan peninsula by ~41.6 ka using fire to penetrate and manipulate forests, an approach possibly used in the first migrations across the last glacial landscape of Sahul.

Complaining as a face-threatening act: A look into AI complaints

This paper aims to conduct an analysis of the complaints as provided by an AI tool, ChatGPT. First, an overview of complaining speech act as a face-threatening act is provided regarding the multiple ways how it is defined and classified, as well as its relation to directness levels. Second, the complaining-realizing strategies with a wide range of modification devices have been summarized and applied as frameworks for scrutinizing AI complaints. Third, the perusal of the complaints gathered from ChatGPT indicates its general adherence to the three-part structure of complaining set, Buffer, Complaint, and Negotiation. The analysis also recorded the implementation of modification tools to show concerns for the interlocutors’ faces.

To shock or to drill: comparison of intravascular lithotripsy and rotational atherectomy - a meta-analysis

Abstract Background Coronary intravascular lithotripsy (IVL) and rotational atherectomy (RA) are potent tools for calcified plaque modification before stenting. While IVL uses pressure waves to breakup calcium, RA mechanically disrupts calcium to promote vascular compliance. It is unclear if one has superior clinical outcomes than the other for calcified coronary lesion preparation before percutaneous coronary intervention (PCI). Methods All relevant studies from PubMed and Embase were included with patients (age >18 years) undergoing PCI for calcified coronary plaque treated with either IVL or RA. All-cause mortality, target lesion revascularization (TLR), and major adverse cardiovascular events (MACE) (mortality, myocardial infarction, TLR, stroke, and in-stent thrombosis) were assessed. The pooled odds ratio (OR) for binary outcomes was computed using the Mantel-Haenszel method. Random effects model was used with 95% confidence interval (CI) for statistical significance. Heterogeneity was marked by Higgins I-squared (I2) statistic. Analysis was done on Comprehensive R Archive Network in R (CRAN-R) software. Results Three studies were included (IVL n=351, RA n=118). Compared to RA, IVL had similar all-cause mortality (OR 1.0, CI 0.3-3.0, p 0.9), TLR (OR 2.1, CI 0.26-18.2, p 0.4), and MACE (OR 2.0, CI 0.9-4.1, p 0.6). There was a trend towards favorable outcomes for RA, although statistically insignificant (figure 1). Conclusion For patients undergoing PCI for calcified coronary lesions, IVL and RA demonstrated comparable clinical outcomes including all-cause mortality. Large-scale prospective studies are needed to understand the differences in clinical outcomes.

Chemoenzymatic Synthesis of Plant‐Derived Kavalactone Natural Products by Dynamic Resolution Using a Biosynthetic O‐Methyltransferase Tailoring Enzyme

AbstractBiosynthetic enzymes have enormous potential for the chemoenzymatic synthesis of natural products and other bioactive compounds. Methyltransferases are promising tools for the selective enzymatic modification of complex structures. This paper describes the production, purification and biochemical characterization of the O‐methyltransferase JerF, which catalyzes unique 4‐methoxy‐5,6‐dihydropyranone formation in jerangolid A biosynthesis. Isolation problems had hitherto prevented detailed studies on JerF and were solved by the fusion to maltose‐binding protein. The differentiation of JerF between styryl‐substituted dihydropyrandion enantiomers was investigated. In combination with a spontaneous racemization occurring with this type of substrates, a new enzymatic dynamic kinetic resolution was observed, which was used for the enantioselective chemoenzymatic synthesis of kavalactone natural products and new derivatives. In combination with an HMT‐based SAM regeneration system, (+)‐kavain, (+)‐11,12‐dimethoxykavain and (+)‐12‐fluorokavain were prepared in 3–4 steps on a 100 μmol scale with overall yields of 37–57 % and ees of 70–86 %. A mutational study based on an AlphaFold 2 model provided indications for active site residues with an influence on the performance of the enzyme that could be targeted for engineering in the future. This example illustrates how the exceptional enzymatic activities and specificities of biosynthetic enzymes can be exploited for the development of new synthesis approaches.

Late-Stage Modification of Peptides with Maleimides through Palladium-Catalyzed β-C(sp3)-H Alkylation.

Transition-metal-catalyzed C-H activation has proven to be a powerful tool for the late-stage modification of peptides. We herein report a method for site-selective alkylation of peptides with maleimides through Pd-catalyzed β-C(sp3)-H activation. In this protocol, the methionine residues within peptides serve as the directing groups, which circumvented the preinstallation and subsequent removal of the directing groups. This chemistry exhibited broad substrate scope and can be utilized for peptide ligation.

Application of Sortase-Mediated Ligation for the Synthesis of Block Copolymers and Protein-Polymer Conjugates.

Sortase-mediated ligation (SML) has become a powerful tool for site-specific protein modification. However, sortaseA (SrtA) suffers from low catalytic efficiency and mediates an equilibrium reaction. Therefore, ligations with large macromolecules may be challenging. Here, the synthesis of polymeric building blocks for sortase-mediated ligation constituting peptide-polymers with either the recognition sequence for sortaseA (LPX1TGX2) or its nucleophilic counterpart (Gx) is demonstrated. The peptide-polymers are synthesized by solid-phase peptide synthesis followed by photo-iniferter (PI) reversible addition-fragmentation chain-transfer (RAFT) polymerization of various monomers. The building blocks are subsequently utilized to investigate possibilities and limitations when using macromolecules in SML. In particular, diblock copolymers are obtained even when using the orthogonal building blocks in equimolar ratio by exploiting a technique to shift the reaction equilibrium. However, ligations of two polymers can not be achieved when the degree of polymerization exceeds 100. Subsequently, C-terminal protein-polymer conjugates are synthesized. Several polymers are utilized that can replace the omnipresent polyethylene glycol (PEG) in future therapeutics. The conjugation is exemplified with a nanobody that is known for efficient neutralization of SARS-CoV-2. The study demonstrates a universal approach to polymer-LPX1TGX2 and Gx-polymer building blocks and gives insight into their application in SML.

Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated sequence (CRISPR/Cas) system is a cutting-edge genome-editing tool employed to explore the functions of normal and disease-related genes. The CRISPR/Cas system has a remarkable diversity in the composition and architecture of genomic loci and Cas protein sequences. Owing to its excellent efficiency and specificity, this system adds an outstanding dimension to biomedical research on genetic manipulation of eukaryotic cells. However, safe, efficient, and specific delivery of this system to target cells and tissues and their off-target effects are considered critical bottlenecks for the therapeutic applications. Recently discovered anti-CRISPR proteins (Acr) play a significant role in limiting the effects of this system. Acrs are relatively small proteins that are highly specific to CRISPR variants and exhibit remarkable structural diversity. The in silico approaches, crystallography, and cryo-electron microscopy play significant roles in elucidating the mechanisms of action of Acrs. Acrs block the CRISPR/Cas system mainly by employing four mechanisms: CRISPR/Cas complex assembly interruption, target-binding interference, target cleavage prevention, and degradation of cyclic oligonucleotide signaling molecules. Engineered CRISPR/Cas systems are frequently used in gene therapy, diagnostics, and functional genomics. Understanding the molecular mechanisms underlying Acr action may help in the safe and effective use of CRISPR/Cas tools for genetic modification, particularly in the context of medicine. Thus, attempts to regulate prokaryotic CRISPR/Cas surveillance complexes will advance the development of antimicrobial drugs and treatment of human diseases. In this review, recent updates on CRISPR/Cas systems, especially CRISPR/Cas9 and Acrs, and their novel mechanistic insights are elaborated. In addition, the role of Acrs in the novel applications of CRISPP/Cas biotechnology for precise genome editing and other applications is discussed.

Recent advances of CRISPR-based genome editing for enhancing staple crops.

An increasing population, climate change, and diminishing natural resources present severe threats to global food security, with traditional breeding and genetic engineering methods often falling short in addressing these rapidly evolving challenges. CRISPR/Cas systems have emerged as revolutionary tools for precise genetic modifications in crops, offering significant advancements in resilience, yield, and nutritional value, particularly in staple crops like rice and maize. This review highlights the transformative potential of CRISPR/Cas technology, emphasizing recent innovations such as prime and base editing, and the development of novel CRISPR-associated proteins, which have significantly improved the specificity, efficiency, and scope of genome editing in agriculture. These advancements enable targeted genetic modifications that enhance tolerance to abiotic stresses as well as biotic stresses. Additionally, CRISPR/Cas plays a crucial role in improving crop yield and quality by enhancing photosynthetic efficiency, nutrient uptake, and resistance to lodging, while also improving taste, texture, shelf life, and nutritional content through biofortification. Despite challenges such as off-target effects, the need for more efficient delivery methods, and ethical and regulatory concerns, the review underscores the importance of CRISPR/Cas in addressing global food security and sustainability challenges. It calls for continued research and integration of CRISPR with other emerging technologies like nanotechnology, synthetic biology, and machine learning to fully realize its potential in developing resilient, productive, and sustainable agricultural systems.

Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum

BackgroundSclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies.ResultsHerein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1.ConclusionsOur findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.Graphical

Investigation of Anti-inflammatory and Antioxidant Activities of Promising 1,4,5-Trisubstituted Pyrazoles Derivatives of Chalcone Ditosylates

Patients suffering from chronic pain and inflammatory disorders need novel COX-2 inhibitors to be developed with minimum toxicity to the kidneys, heart, and gastrointestinal tract and with excellent anti-inflammatory activity. The current study centers on an array of 1,4,5-trisubstituted pyrazoles produced via the reaction of ditosylates of chalcones with hydrochloride salt of phenylhydrazine. Chalcone was reacted with HTIB to produce a variety of derivatives of α, β chalcone ditosylates. Phenylhydrazine hydrochloride treatment of these chalcone ditosylates produced distinct 1,4,5-trisubstituted pyrazoles. The conversion process is mediated by 1,2-aryl migrations. IR, 1NMR, and elemental analysis were used to characterize the compounds after they had been purified by recrystallization. Using ascorbic acid as a reference, the DPPH (1,1-diphenyl-2-picrylhydrazyl) technique was used to assess the compounds' in vitro antioxidant activity. The paw edema technique caused by Carrageenan was utilized to assess the compounds' in vivo anti-inflammatory properties. The standard medication used was diclofenac sodium. A plethysmograph was used to measure the volume of the rats' paws. When compared to the standard, the compounds V5D5PH5 and V7D7PH7 showed modest antioxidant activity. When the synthetic pyrazoles were examined for their in vivo anti-inflammatory properties, substances V4D4PH4 and V7D7PH7 outperformed the reference. Here, we attempted to create new, safe, and effective drugs for the treatment of inflammatory disorders and pain by utilizing synthetic pyrazole moiety derivatives. Simple experimentation is used in the proposed study to improve pharmacological activity and yields. In the near future, chalcone ditosylate derivatives will be a powerful tool for selective modification.

MetSCORE: a molecular metric to evaluate the risk of metabolic syndrome based on serum NMR metabolomics

BackgroundMetabolic syndrome (MetS) is a cluster of medical conditions and risk factors correlating with insulin resistance that increase the risk of developing cardiometabolic health problems. The specific criteria for diagnosing MetS vary among different medical organizations but are typically based on the evaluation of abdominal obesity, high blood pressure, hyperglycemia, and dyslipidemia. A unique, quantitative and independent estimation of the risk of MetS based only on quantitative biomarkers is highly desirable for the comparison between patients and to study the individual progression of the disease in a quantitative manner.MethodsWe used NMR-based metabolomics on a large cohort of donors (n = 21,323; 37.5% female) to investigate the diagnostic value of serum or serum combined with urine to estimate the MetS risk. Specifically, we have determined 41 circulating metabolites and 112 lipoprotein classes and subclasses in serum samples and this information has been integrated with metabolic profiles extracted from urine samples.ResultsWe have developed MetSCORE, a metabolic model of MetS that combines serum lipoprotein and metabolite information. MetSCORE discriminate patients with MetS (independently identified using the WHO criterium) from general population, with an AUROC of 0.94 (95% CI 0.920–0.952, p < 0.001). MetSCORE is also able to discriminate the intermediate phenotypes, identifying the early risk of MetS in a quantitative way and ranking individuals according to their risk of undergoing MetS (for general population) or according to the severity of the syndrome (for MetS patients).ConclusionsWe believe that MetSCORE may be an insightful tool for early intervention and lifestyle modifications, potentially preventing the aggravation of metabolic syndrome.