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BioRxiv : the Preprint Server For... May 2024display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of...
display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing -(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.
PubMed: 38853888
DOI: 10.1101/2024.05.29.596443 -
Journal of Biomechanics May 2024Degenerative disc disease (DDD), regardless of its phenotype and clinical grade, is widely associated with low back pain (LBP), which remains the single leading cause of...
Degenerative disc disease (DDD), regardless of its phenotype and clinical grade, is widely associated with low back pain (LBP), which remains the single leading cause of disability worldwide. This work provides a quantitative methodology for comparatively investigating artificial IVD degeneration via two popular approaches: enzymatic denaturation and fatigue loading. An in-vitro animal study was used to study the time-dependent responses of forty fresh juvenile porcine thoracic IVDs in conjunction with inverse and forward finite element (FE) simulations. The IVDs were dissected from 6-month-old-juvenile pigs and equally assigned to 5 groups (intact, denatured, low-level, medium-level, high-level fatigue loading). Upon preloading, a sinusoid cyclic load (Peak-to-peak/0.1-to-0.8 MPa) was applied (0.01-10 Hz), and dynamic-mechanical-analyses (DMA) was performed. The DMA outcomes were integrated with a robust meta-model analysis to quantify the poroelastic IVD characteristics, while specimen-specific FE models were developed to study the detailed responses. The results demonstrated that enzymatic denaturation had a more significantly pronounced effect on the resistive strength and shock attenuation capabilities of the intervertebral discs. This can be attributed to the simultaneous disruption of the collagen fibers and water-proteoglycan bonds induced by trypsin digestion. Fatigue loading, on the other hand, primarily influenced the disc's resistance to deformation in a frequency-dependent pattern, where alterations were most noticeable at low loading frequencies. This study confirms the intricate interplay between the biochemical changes induced by enzymatic processes and the mechanical behavior stemming from fatigue loading, suggesting the need for a comprehensive approach to closely mimic the interrelated multifaceted processes of human disc degeneration.
PubMed: 38852480
DOI: 10.1016/j.jbiomech.2024.112159 -
Ultrasound in Medicine & Biology Aug 2024Ultrasound-triggered bubble-mediated local drug delivery has shown potential to increase therapeutic efficacy and reduce systemic side effects, by loading drugs into the...
OBJECTIVE
Ultrasound-triggered bubble-mediated local drug delivery has shown potential to increase therapeutic efficacy and reduce systemic side effects, by loading drugs into the microbubble shell and triggering delivery of the payload on demand using ultrasound. Understanding the behavior of the microbubbles in response to ultrasound is crucial for efficient and controlled release.
METHODS
In this work, the response of microbubbles with a coating consisting of poly(2-ethyl-butyl cyanoacrylate) (PEBCA) nanoparticles and denatured casein was characterized. High-speed recordings were taken of single microbubbles, in both bright field and fluorescence.
RESULTS
The nanoparticle-loaded microbubbles show resonance behavior, but with a large variation in response, revealing a substantial interbubble variation in mechanical shell properties. The probability of shell rupture and the probability of nanoparticle release were found to strongly depend on microbubble size, and the most effective size was inversely proportional to the driving frequency. The probabilities of both rupture and release increased with increasing driving pressure amplitude. Rupture of the microbubble shell occurred after fewer cycles of ultrasound as the driving pressure amplitude or driving frequency was increased.
CONCLUSION
The results highlight the importance of careful selection of the driving frequency, driving pressure amplitude and duration of ultrasound to achieve the most efficient ultrasound-triggered shell rupture and nanoparticle release of protein-and-nanoparticle-stabilized microbubbles.
Topics: Microbubbles; Nanoparticles; Drug Delivery Systems; Drug Liberation; Enbucrilate; Caseins; Proteins
PubMed: 38851940
DOI: 10.1016/j.ultrasmedbio.2024.03.011 -
BMC Oral Health Jun 2024This study investigated the effect of carbodiimide (EDC) combined with Clearfil SE self-etch adhesive on the shear bond strength (SBS), crosslinking degree, denaturation...
BACKGROUND
This study investigated the effect of carbodiimide (EDC) combined with Clearfil SE self-etch adhesive on the shear bond strength (SBS), crosslinking degree, denaturation temperature, and enzyme activity of dentin in vitro.
MATERIALS AND METHODS
Collected human sound third molars were randomly divided into different groups with or without EDC treatment (0.01-1 M). The specimens (n = 16)were stored for 24 h (immediate) or 12 months (aging) before testing the SBS. Fine dentin powder was obtained and treated with the same solutions. Then the crosslinking degree, denaturation temperature (Td), and enzyme activity were tested. Statistical analysis was performed using a one-way analysis of variance (ANOVA) to compare the differences of data between groups (α = 0.05).
RESULTS
There was a significant drop in immediate SBS and more adhesive fracture of 1.0 M EDC group, while there were no significant differences among the other groups. SEM showed a homogeneous interface under all treatments. After 12 months of aging, the SBS significantly decreased. Less decreases of SBS in the 0.3 and 0.5 M groups were found. Due to thermal and enzymatical properties consideration, the 0.3 and 0.5 M treatments also showed higher cross-link degree and Td with lower enzyme activity.
CONCLUSION
0.3 and 0.5 M EDC may be favorable for delaying the aging of self-etch bond strength for 12 months. But it is still needed thoroughly study.
Topics: Humans; Carbodiimides; Resin Cements; Shear Strength; Materials Testing; Dentin; Microscopy, Electron, Scanning; Dentin-Bonding Agents; Dental Stress Analysis; Cross-Linking Reagents; Dental Bonding; In Vitro Techniques; Acid Etching, Dental; Molar, Third; Temperature; Time Factors; Surface Properties
PubMed: 38849778
DOI: 10.1186/s12903-024-04415-2 -
Current Health Sciences Journal 2024Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed...
Inflammation and the injuries produced by free radicals are interconnected and influence each other. The underlying mechanisms of inflammation are partially attributed to the release of free radicals by immune cells, prooxidants that can also cause protein alteration. This study was performed in order to assess the potential anti-inflammatory effect of two bee venom samples harvested from Apis mellifera. Free radical scavenging capacity was investigated using DPPH and ABTS.+ tests and protective effect on proteins through the inhibitory activity on thermal denaturation of albumin.
PubMed: 38846469
DOI: 10.12865/CHSJ.50.01.11 -
Microbes and Environments 2024All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells... (Review)
Review
All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells by denaturing the DNA double helix, causing chemical damage to DNA, and increasing the random thermal motion of chromosomes. Thermophiles, predominantly classified as bacteria or archaea, exhibit an exceptional capacity to mitigate these detrimental effects and prosper under extreme thermal conditions, with some species tolerating temperatures higher than 100°C. Their genomes are mainly characterized by the presence of reverse gyrase, a unique topoisomerase that introduces positive supercoils into DNA. This enzyme has been suggested to maintain the genome integrity of thermophiles by limiting DNA melting and mediating DNA repair. Previous studies provided significant insights into the mechanisms by which NAPs, histones, SMC superfamily proteins, and polyamines affect the 3D genomes of thermophiles across different scales. Here, I discuss current knowledge of the genome organization in thermophiles and pertinent research questions for future investigations.
Topics: Archaea; Bacteria; Genome, Bacterial; Genome, Archaeal; Hot Temperature; DNA Topoisomerases, Type I; DNA Repair
PubMed: 38839371
DOI: 10.1264/jsme2.ME23087 -
Scientific Reports Jun 2024Previous studies showed that Australian wheat cultivars Janz and Sunco carry leaf rust and stem rust resistance genes Lr24 and Sr24 derived from Thinopyrum ponticum...
Previous studies showed that Australian wheat cultivars Janz and Sunco carry leaf rust and stem rust resistance genes Lr24 and Sr24 derived from Thinopyrum ponticum chromosome arm 3AgL. However, the size of the alien segments carrying Lr24 and Sr24 in the lines were not determined. In this study, we used non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and PCR-based landmark unique gene (PLUG) markers to visualize the alien segments in Janz and Sunco, and further compared them with the segments in US cultivars Agent and Amigo. The fraction length (FL) of the alien translocation in Agent was 0.70-1.00, whereas those in Janz, Sunco, and Amigo were smaller, at FL 0.85-1.00. It was deduced that the alien gene R encoding for red grain color and rust resistance genes Lr24 and Sr24 on chromosome arm 3AgL were in bins of FL 0.70-0.85 and 0.85-1.00, respectively. We retrieved and extracted nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor genes corresponding to the region of Lr24 and Sr24 on chromosomes 3E, and 3J, 3J and 3St from the reference genome sequences of Th. elongatum and Th. intermedium, respectively. A set of molecular markers developed for Lr24 and Sr24 from those extracted NBS-LRR genes will provide valuable information for fine mapping and cloning of these genes.
Topics: Triticum; Disease Resistance; Plant Diseases; Chromosomes, Plant; Genes, Plant; In Situ Hybridization, Fluorescence; Basidiomycota; Chromosome Mapping
PubMed: 38834653
DOI: 10.1038/s41598-024-63835-w -
RSC Advances May 2024An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of...
An operationally simple one-pot three-component and convenient synthesis method for a series of diverse purine analogues of 5-amino-7-(substituted)--(4-sulfamoylphenyl)-4,7-dihydro-[1,2,4]-triazolo[1,5-][1,3,5]triazine-2-carboxamide derivatives generated the reaction of 2-hydrazinyl--(4-sulfamoylphenyl)-2-thioxoacetamide, cyanoguanidine and a variety of aldehydes was achieved under green conditions. This experiment was conducted to evaluate the anti-inflammatory effect of the newly synthesized compounds using indomethacin as a reference medication; all compounds were tested for anti-inflammatory activity using the inhibition of albumin denaturation, RBC hemolysis technique and COX inhibition assay. The results showed that all evaluated compounds exhibited significant anti-inflammatory efficacy leading to excellently effective RBC membrane stabilization, inhibition of protein denaturation, and inhibition of COX enzymes when compared to those of indomethacin. At concentrations of 50, 100, 200, and 300 μg ml, these compounds decreased COX-1 and COX-2 activities more than indomethacin and have IC values in the range of 40.04-87.29 μg ml for COX-1 and 27.76-42.3 μg ml for COX-2 while indomethacin showed IC = 91.57 for COX-1 and 42.66 μg ml for COX-2. The anti-inflammatory findings show the need for more investigation to define the properties underlying the evaluated compounds' anti-inflammatory abilities. The enzyme cyclooxygenase-2 (COX 2) (PDB ID: 5IKT) was docked with ten synthetic substances. With docking scores () of -8.82, -7.82, and -7.76 kcal mol, 7-furan triazolo-triazine (4), 7-(2-hydroxy phenyl) triazolo-triazine (11), and 7-(4-dimethylamino phenyl) triazolo-triazine (12) had the greatest binding affinities, respectively. Therefore, these substances have COX-2 (PDB ID: 5IKT) inhibitory capabilities and hence may be investigated for COX 2 targeting development. Furthermore, both the top-ranked compounds (4 and 11) and the standard indomethacin were subjected to DFT analysis. The HOMO - LUMO energy difference (Δ) of the mentioned compounds was found to be less than that of indomethacin.
PubMed: 38832248
DOI: 10.1039/d4ra02970d -
RSC Advances May 2024Polysaccharides are considered to be ideal green raw materials for enhancing biocompatibility and dispersion effects of nanoparticles. In this study, we coated and...
Polysaccharides are considered to be ideal green raw materials for enhancing biocompatibility and dispersion effects of nanoparticles. In this study, we coated and dispersed ZnO nanoparticles (NPs) using the denaturation-renaturation process of a triple helix glucan lentinan (LNT), induced by changes in pH value within the reaction system. Various ZnO/LNT composites with different particle sizes and crystal morphologies were prepared and characterized. The results demonstrated that renatured LNT (r-LNT) effectively encapsulated the {101̄0} crystal planes of ZnO, preventing crystal growth during the renaturation process and resulting in smaller, uniformly dispersed nanoparticles. Among the samples, ZnO/r-LNT-2 exhibited significantly higher antimicrobial activity, and it had a certain inhibitory effect on various plant pathogens. It also displayed the highest inhibitory effect against , with a minimum inhibitory concentration (MIC) of up to 8 μg mL. Consistently, ZnO/r-LNT-2 generated the highest amount of reactive oxygen species (ROS), thus exhibiting the most effective antimicrobial activity. However, excessive introduction of the dispersant LNT may reduce these activities. This study provides a foundation for further exploring the detailed mechanism of ROS generation catalyzed by ZnO and for harnessing the full potential of this type of antimicrobial agent.
PubMed: 38832239
DOI: 10.1039/d4ra01590h