Conjugation Method Research Articles

New Method for Solving the Inverse Thermal Conduction Problem (θ-Scheme Combined with CG Method under Strong Wolfe Line Search)

Most thermal researchers have solved thermal conduction problems (inverse or direct) using several different methods. These include the usual discretization methods, conventional and special estimation methods, in addition to simple synchronous gradient methods such as finite elements, including finite and special quantitative methods. Quantities found through the finite difference methods, i.e., explicit, implicit or Crank–Nicolson scheme method, have also been adopted. These methods offer many disadvantages, depending on the different cases; when the solutions converge, limited range stability conditions. Accordingly, in this paper, a new general outline of the thermal conduction phenomenon, called (θ-scheme), as well as a gradient conjugate method that includes strong Wolfe conditions has been used. This approach is the most useful, both because of its accuracy (16 decimal points of importance) and the speed of its solutions and convergence; by addressing unfavorable adverse problems and stability conditions, it can also have wide applications. In this paper, we applied two approaches for the control of the boundary conditions: constant and variable. The θ-scheme method has rarely been used in the thermal field, though it is unconditionally more stable for θ∈ [0.5, 1]. The simulation was carried out using Matlab software.

Recent Advances in Antibody-drug Conjugates Produced Using Chemical Conjugation Technology

To date, more than 10 antibody-drug conjugates (ADCs) have been approved by the U.S. Food and Drug Administration (FDA) and contribute to the medical community. Interestingly, all of them are manufactured using chemical conjugation techniques. This demonstrates that chemical conjugation is the primary approach to ADC manufacturing. Compared to other conjugation techniques (e.g., genetic engineering or enzymatic), chemical conjugation has the advantage of a relatively simple chemistry, manufacturing, and control (CMC) for ADC, prompting pharmaceutical companies to choose this technique for ADC projects. To better understand this advantage, this article provides an overview of established full chemical conjugation methods that have already been applied in the scale-up phase for ADC production.

The Future of Antibody Drug Conjugation by Comparing Various Methods of Site-Specific Conjugation.

The field of oncology is continuously seeking to find effective treatment therapies with limited side effects. Antibody-drug conjugates (ADCs) are a promising class of cancer therapies that have been shown to be effective with limited side effects. Although promising, these therapies experience shortcomings, such as the stability and reproducibility of current conjugation methods. Historically, ADCs have been produced by stochastic conjugation methods; however, new methods of site-specific conjugation have evolved to mitigate current ADC shortcomings. In this article, we highlight the success of ADCs as well as some of their challenges. We also highlight the shortcomings of stochastic conjugation and explore the various site-specific conjugation methods and their advantages over stochastic conjugation.

Exosomes: Recent Advances and Challenges as Targeted Therapeutic Delivery Vesicles

The exosome is a naturally derived nanostructured lipid vesicle that ranges from 40-100 nm in size and is utilized to transport drugs, and biological macromolecules, including therapeutic RNA and proteins. It is a membrane vesicle actively released by cells to transport cellular components with a purpose for biological events. The conventional isolation technique has several drawbacks, including low integrity, low purity, long processing time, and sample preparation. Therefore, microfluidic technologies are more widely accepted for the isolation of pure exosomes, but due to cost and expertise requirements, this technology is also facing challenges. The bioconjugation of small and macro-molecules to the surface of exosomes is a very interesting and emerging approach for achieving the specific target, therapeutic purpose, in vivo imaging, and many more. Although emerging strategies resolve a few challenges, exosomes are still unexplored complex nano-vesicles with excellent properties. This review has briefly elaborated on contemporary isolation techniques and loading approaches. We have also discussed the surface-modified exosomes by different conjugation methods and their applications as targeted drug delivery vesicles. The challenges associated with the exosomes, patents, and clinical investigations are the main highlight of this review.

Construction of a conjugation method for the transformation of Cobetia sp. IU180733JP01 (5-11-6-3) that accumulates poly(3-hydroxybutyrate) from seaweeds.

Cobetia bacteria are considered useful hosts for industrial applications owing to their fast growth, high cell density, and halophilicity. Here, we constructed an efficient conjugation method to obtain Cobetia sp. IU180733JP01 (5-11-6-3) transformants, which can produce bioplastics from alginate or seaweed waste. Lysogeny Broth medium containing 2% NaCl was used to co-cultivate the 5-11-6-3 strain (plasmid recipient) with Escherichia coli S17-1 (plasmid donor). Transformants with the highest conjugation efficiency [(2.92 ± 1.37) × 10-3] were obtained at a donor:recipient cell number ratio of 5:1. This is the first study reporting the creation of recombinant strains in the genus Cobetia. This method will contribute to creating a platform strain for the production of bioplastics and other useful materials from marine biomass.

Mass Spectrometry-Based Tissue Imaging of the Tumor Microenvironment.

Multiplex ion beam imaging (MIBI) and imaging mass cytometry (IMC) enable highly multiplexed antibody (40+) staining of frozen or formalin fixed, paraffin-embedded (FFPE) human or murine tissues through detection of metal ions liberated from primary antibodies by time-of-flight mass spectrometry (TOF). These methods make detection of more than 50 targets theoretically possible while maintaining spatial orientation. As such, they are ideal tools to identify the multiple immune, epithelial, and stromal cell subsets in the tumor microenvironment and to characterize spatial relationships and tumor-immune status in either murine models or human samples. This chapter summarizes methods for antibody conjugation and validation, staining, and preliminary data collection using IMC or MIBI in both human and mouse pancreatic adenocarcinoma samples. These protocols are intended to facilitate use of these complex platforms in not only tissue-based tumor immunology studies but also tissue-based oncology or immunology studies more broadly.

Potato Virus X Inactivation and Characterization.

The nucleoprotein components of plant viruses self-assemble into monodisperse, nanoscale structures with a high degree of symmetry and polyvalency. Of particular interest are the filamentous plant viruses which provide uniform high aspect ratio nanostructures-such structures remain challenging to obtain using purely synthetic approaches. Potato virus X (PVX) has drawn interest by the materials science community because of its filamentous structure measuring 515×13nm; and both genetic engineering and chemical conjugation methods have been reported to impart new functionalities and develop PVX-based nanomaterials for applications in the health and materials sector. Toward environmentally safe materials-i.e., materials that are not infectious toward crops, such as potato, we reported methods to inactivate PVX. In this chapter, we describe the three methods to inactivate PVX and render it non-infectious toward plants, while maintaining structure and function.

The enhancement of enzyme cascading via tetrahedral DNA framework modification.

Enzyme clustering is widely used in many organisms to increase the catalytic efficiency of cascade reactions. Inspired by nature, organizing enzymes within a cascade reaction also draws much attention in both basic research and industrial processes. An important step for organizing enzymes precisely in vitro is enzyme modification. However, modifying enzymes without sacrificing their activity remains challenging until now. For example, labeling enzymes with DNA, one of the well-established enzyme modification methods, has been shown to significantly reduce the enzymatic activity. Herein we report an enzyme conjugation method that can rescue the reduction of enzymatic activity caused by DNA labeling. We demonstrate that immobilizing DNA-modified enzymes on the vertex of TDNs (tetrahedral DNA nanostructures) enhances the enzymatic activity compared with their unmodified counterparts. Using this strategy, we have further developed an ultra-sensitive and high-throughput electrochemical biosensor for sarcosine detection, which holds great promise for prostate cancer screening.

Anti-restriction protein ArdA promotes clinical Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae spread and its molecular mechanism

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) has spread worldwide and has become a major threat to public health. The restriction modification system provides an innate defence of bacteria against plasmids or transposons, while many different types of plasmid encoding the anti-restriction protein ArdA can specifically affect the restriction activity in bacteria. To detect the codistribution of ArdA and blaKPC-2 plasmids in KPC-KP and explore the molecular mechanism of ArdA promoting KPC-KP spread. We collected 65 clinical CRKP isolates from Ningbo, China, and 68 cases of plasmid complete sequences in GenBank to determine the prevalence of ArdA gene on the K. pneumoniae blaKPC-2 plasmid. The anti-restriction function of ArdA in promoting horizontal gene transfer (HGT) was verified by transformation, conjugation and transduction methods, and the pull-down experiment was used to investigate the molecular mechanism of ArdA protein in vitro. We found that ArdA was widely distributed in KPC-KP in 100% of cases, which was detected in 0% of drug susceptible K. pneumoniae, and the plasmids containing the ArdA gene in 90% of the 30 cases randomly retrieved from the database. We also verified that ArdA has a good anti-restriction function (P < 0.05) through two aspects of HGT (transformation, transduction), and explored the non-occurrence interaction of ArdA and the hsdM subunit protein of EcoKI enzyme from the perspective of protein molecules. These findings suggest that the coexistence advantage of ArdA with the blaKPC-2 plasmids may provide KPC-producing K. pneumoniae with a very efficient evasion of the restriction of type I systems, which not only favours ArdA-containing mobile genetic elements in the same species HGT between bacteria also facilitates HGT between other bacterial species.

Heuristic Potential of Library Bibliographic Classification

In the era when knowledge and information have become a strategic resource, theoretical and practical issues of building classification systems have seen an increased interest. The study of various library classifications, as well as their comparative analysis, allows us to identify local (and/or global) trends in the development of theory and practice of library and information activities. The multidimensional vision of heuristic potential as a complex of possibilities for finding effective new tools and techniques for solving complex problems has determined the scope and features of the research method. The purpose of the article is to determine the methodological mechanisms which can help expanding the capabilities of Library Bibliographical Classification (LBC) in solving cognitive, research and creative tasks. Realization of this goal was carried out on the basis of historical and methodological reconstruction method, rarely used at present. The object of historical and methodological reconstruction is the technology of knowledge organization. Subject scientific interest is focused on the study of a set of gnoseological tools and techniques that facilitate and simplify the solution of problems of library classification. Basing on the scientific publications of the staff of the Research Centre for the Development of LBC of the Russian State Library, as well as texts of Medium Schedules (2001—2019, issues 1—8), Abridged Schedules (2021) and Schedules for children’s and school libraries (2016), the authors present the original methodological base for updating the LBC of the Russian State Library. The paper reveals that the core of this methodological base is the method of scientific conjugation. The authors test the hypothesis of transformation of the LBC from the system of organizing the existing knowledge into a system of producing new knowledge with the expanded functionality. In addition to the traditional applied functions of the LBC — organizational, reference, information, methodological — classification also performs cognitive, enlightening, educational and research functions. The heuristic potential of the LBC is revealed through the knowledge and experience in different subject areas.

Ultra-broadband polarization-independent omnidirectional reflectors via dielectric-reversal quasicrystal heterostructure multilayer films

Since quasicrystals provide additional freedom to expand photonic bandgap, dielectric quasiperiodic sequences of broadbands become crucial for implementing low-loss omnidirectional reflectors or filters. The current major challenge is to find an elegant scheme to construct angle-insensitive multilayer structures with fewer layer numbers. In this work, we create an appealing quasiperiodic ordering of the dielectric-reversal Fibonacci sequence to optimize the properties of omnidirectional reflectors. We have concatenated it to a periodic sequence to construct the omnidirectional reflector of hybrid multilayers with thin thickness and reflectivity above 99% over a visible scope from 436 to 559 nm. An ultrabroad polarization-independent omnidirectional bandgap of the relative bandwidth of 39.8% has been achieved by cascading two dielectric-reversal ordering sequences. The self-similarity of Fibonacci multilayer structures along with this characteristic quasiperiodic order guarantees the existence of perfect omnidirectional reflections at any scale. This ordering strategy of dielectric-reverse essentially differs from previous conjugate and recursion methods, which will significantly enrich the variety of quasiperiodic orders in theory and applied science domains.

Some quantitative one-sided weighted estimates

In this paper we provide some quantitative one-sided estimates that recover the dependences in the classical setting. Among them we provide estimates for the one-sided maximal function in Lorentz spaces and we show that the conjugation method for commutators works as well in this setting.

Functional improvements in β-conglycinin by edible bioconjugation with carboxymethyl dextran.

β-Conglycinin was conjugated with carboxymethyl dextran (CMD) by the Maillard reaction to improve its function. The β-conglycinin-CMD conjugate was purified by dialysis. Conjugation was confirmed by SDS-PAGE with CBB and PAS staining. Composition of the β-conglycinin-CMD was β-conglycinin:CMD = 1:2.7 (molar ratio) which was confirmed by BCA method and phenol sulfuric acid method. Solubility of β-conglycinin in the range of pH 2.0-7.0 was much improved by conjugation with CMD. Emulsifying property of β-conglycinin at pH 7 and in presence of salt was improved by conjugation with CMD. Immunogenicity of β-conglycinin was reduced by conjugation with CMD. Conjugation method performed in this study was considered to be valuable in that it can be used in food processing.

Production of hemp protein isolate-polyphenol conjugates through ultrasound and alkali treatment methods and their characterization

Two easy-to-implement methods, i.e., ultrasound and alkali treatment, were used to produce conjugates of hemp protein isolate (HPI) and polyphenols (gallic acid and catechin). The effect of these two methods of conjugation on the degree of conjugation (free amino and sulfhydryl group contents), particle size, charge tensity, secondary and tertiary structures, surface hydrophobicity, solubility and emulsifying properties, antioxidant activities of the resulting conjugates were determined. The effect of conjugation on the storage, temperature, and salt stability was evaluated. The results showed that alkali treatment induced more polyphenols to conjugate with HPI than ultrasound treatment. The conjugation significantly improved the solubility, emulsifying properties of HPI, especially in the case of HPI-gallic acid conjugate prepared through ultrasound treatment. The improvement in technofunctional properties correlated with unfolding of molecules, reduced particle size, surface hydrophobicity, interfacial tension, and increased charge intensity. The conjugation of HPI with polyphenols improved its antioxidant activities. Emulsions stabilized by conjugates also exhibited higher storage, temperature and salt stability. These findings indicate that these two easy-to-implement conjugation methods can be readily used to develop HPI-polyphenol conjugates and use them as new class of emulsifier.

Readily Accessible Strained Difunctionalized trans-Cyclooctenes with Fast Click and Release Capabilities.

The click reaction between a functionalized trans-cyclooctene (TCO) and a tetrazine (Tz) is a compelling method for bioorthogonal conjugation in combination with payload releasing capabilities. However, the synthesis of difunctionalized TCOs remains challenging. As a result, these compounds are poorly accessible, which impedes the development of novel applications. In this work, the scalable and accessible synthesis of a new bioorthogonal difunctionalized TCO is reported in only four single selective high yielding steps starting from commercially available compounds. The TCO-Tz click reaction was assessed and revealed excellent kinetic rates and subsequently payload release was shown with various functionalized derivatives. Tetrazine triggered release of carbonate and carbamate payloads was demonstrated up to 100 % release efficiency and local drug release was shown in a cellular toxicity study which revealed a >20-fold increase in cytotoxicity.

Conjugation Mechanism for Pneumococcal Glycoconjugate Vaccines: Classic and Emerging Methods.

Licensed glycoconjugate vaccines are generally prepared using native or sized polysaccharides coupled to a carrier protein through random linkages along the polysaccharide chain. These polysaccharides must be chemically modified before covalent linking to a carrier protein in order to obtain a more defined polysaccharide structure that leads to a more rational design and safer vaccines. There are classic and new methods for site-selective glycopolysaccharide conjugation, either chemical or enzymatic modification of the polysaccharide length or of specific amino acid residues of the protein carrier. Here, we discuss the state of the art and the advancement of conjugation of S. pneumoniae glycoconjugate vaccines based on pneumococcal capsular polysaccharides to improve existing vaccines.

PafS Containing GGDEF-Domain Regulates Life Activities of Pseudomonas glycinae MS82.

Cyclic dimeric guanosine monophosphate (c-di-GMP) is synthesized by diguanylate cyclase (DGC) with the GGDEF domain. As a ubiquitous bacterial second messenger, it regulates diverse life-activity phenotypes in some bacteria. Although 38 genes encoding GGDEF-domain-containing proteins have been identified in the genome of the Pseudomonas glycinae strain MS82, whether c-di-GMP functions as a facilitator or repressor of life-activity phenotypes is poorly understood. In this study, one of the 38 genes containing a GGDEF domain in MS82, PafS was investigated to explore its regulatory function in bacterial life activities. The PafS-deletion mutant ΔPafS and reversion mutant PafS-comp were constructed by the method of biparental conjugation and homologous recombination. The life activities of the mutants, such as antifungal activity, biofilm formation ability, polysaccharide content, and motor behavior, were explored. The results showed that all life-activity phenotypes were significantly reduced after knocking out PafS, whereas all were significantly restored to a similar level to that of MS82 after the complementation of PafS. These results suggested that PafS plays an important role in the regulation of a range of cellular activities by c-di-GMP in P. glycinae MS82.

IUP-BERT: Identification of Umami Peptides Based on BERT Features.

Umami is an important widely-used taste component of food seasoning. Umami peptides are specific structural peptides endowing foods with a favorable umami taste. Laboratory approaches used to identify umami peptides are time-consuming and labor-intensive, which are not feasible for rapid screening. Here, we developed a novel peptide sequence-based umami peptide predictor, namely iUP-BERT, which was based on the deep learning pretrained neural network feature extraction method. After optimization, a single deep representation learning feature encoding method (BERT: bidirectional encoder representations from transformer) in conjugation with the synthetic minority over-sampling technique (SMOTE) and support vector machine (SVM) methods was adopted for model creation to generate predicted probabilistic scores of potential umami peptides. Further extensive empirical experiments on cross-validation and an independent test showed that iUP-BERT outperformed the existing methods with improvements, highlighting its effectiveness and robustness. Finally, an open-access iUP-BERT web server was built. To our knowledge, this is the first efficient sequence-based umami predictor created based on a single deep-learning pretrained neural network feature extraction method. By predicting umami peptides, iUP-BERT can help in further research to improve the palatability of dietary supplements in the future.

Modular Site-Specific Conjugation of Nanobodies Using Two Co-Associating Tags.

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of VHH and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in Escherichia coli in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.

Reduced immunogenicity and endowed antimicrobial activity in β-lactoglobulin by preparing edible bioconjugate.

β-lactoglobulin (BLG) and ε-polylysine (PL) were bound by using microbial transglutaminase. Dextran (Dex) was further conjugated to the BLG-PL conjugate by the Maillard reaction. Confirmation of conjugation was carried out by SDS-PAGE. From the results of isoelectric focusing, it was revealed that the isoelectric point of the BLG-PL conjugate was shifted to the basic side as compared with native BLG. Immunogenicity of BLG in BALB/c mice was lowered by conjugation with PL and further lowered by conjugation with Dex. By conjugation with PL and PL-Dex, antibacterial activity against Staphylococcus aureus was endowed to BLG. Because the conjugation method in this study is a safe method, it is valuable in that it can be applicable to food processing.