Conjugation Process Research Articles

Immunogenicity of Current and Next-Generation Pneumococcal Conjugate Vaccines in Children: Current Challenges and Upcoming Opportunities.

Global use of pneumococcal conjugate vaccines (PCVs) with increasingly broader serotype coverage has helped to reduce the burden of pneumococcal disease in children and adults. In clinical studies comparing PCVs, higher-valency PCVs have met noninferiority criteria (based on immunoglobulin G geometric mean concentrations and response rates) for most shared serotypes. A numeric trend of declining immunogenicity against shared serotypes with higher-valency PCVs has also been observed; however, the clinical relevance is uncertain, warranting additional research to evaluate the effectiveness of new vaccines. Novel conjugation processes, carriers, adjuvants, and vaccine platforms are approaches that could help maintain or improve immunogenicity and subsequent vaccine effectiveness while achieving broader protection with increasing valency in pneumococcal vaccines.

Biology System Description Language (BiSDL): a modeling language for the design of multicellular synthetic biological systems

BackgroundThe Biology System Description Language (BiSDL) is an accessible, easy-to-use computational language for multicellular synthetic biology. It allows synthetic biologists to represent spatiality and multi-level cellular dynamics inherent to multicellular designs, filling a gap in the state of the art. Developed for designing and simulating spatial, multicellular synthetic biological systems, BiSDL integrates high-level conceptual design with detailed low-level modeling, fostering collaboration in the Design-Build-Test-Learn cycle. BiSDL descriptions directly compile into Nets-Within-Nets (NWNs) models, offering a unique approach to spatial and hierarchical modeling in biological systems.ResultsBiSDL’s effectiveness is showcased through three case studies on complex multicellular systems: a bacterial consortium, a synthetic morphogen system and a conjugative plasmid transfer process. These studies highlight the BiSDL proficiency in representing spatial interactions and multi-level cellular dynamics. The language facilitates the compilation of conceptual designs into detailed, simulatable models, leveraging the NWNs formalism. This enables intuitive modeling of complex biological systems, making advanced computational tools more accessible to a broader range of researchers.ConclusionsBiSDL represents a significant step forward in computational languages for synthetic biology, providing a sophisticated yet user-friendly tool for designing and simulating complex biological systems with an emphasis on spatiality and cellular dynamics. Its introduction has the potential to transform research and development in synthetic biology, allowing for deeper insights and novel applications in understanding and manipulating multicellular systems.

Enzymatically Triggered Peptide-Lipid Conjugation of Designed Membrane Active Peptides for Controlled Liposomal Release.

Possibilities for controlling the release of pharmaceuticals from liposomal drug delivery systems can enhance their efficacy and reduce their side effects. Membrane-active peptides (MAPs) can be tailored to promote liposomal release when conjugated to lipid head groups using thiol-maleimide chemistry. However, the rapid oxidation of thiols hampers the optimization of such conjugation-dependent release strategies. Here, we demonstrate a de novo designed MAP modified with an enzyme-labile Cys-protection group (phenylacetamidomethyl (Phacm)) that prevents oxidation and facilitates in situ peptide lipidation. Before deprotection, the peptide lacks a defined secondary structure and does not interact with maleimide-functionalized vesicles. After deprotection of Cys using penicillin G acylase (PGA), the peptide adopts an α-helical conformation and triggers rapid release of vesicle content. Both the peptide and PGA concentrations significantly influence the conjugation process and, consequently, the release kinetics. At a PGA concentration of 5 μM the conjugation and release kinetics closely mirror those of fully reduced, unprotected peptides. We anticipate that these findings will enable further refinement of MAP conjugation and release processes, facilitating the development of sophisticated bioresponsive MAP-based liposomal drug delivery systems.

Cylindrospermopsin enhances the conjugative transfer of plasmid-mediated multi-antibiotic resistance genes through glutathione biosynthesis inhibition

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1–100 μg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 μg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.

Abstract 3131: Novel CDH17-targeting antibody-drug conjugate exhibits anti-tumor efficacy in preclinical models of gastrointestinal cancers

Abstract Antibody-drug conjugates (ADC) for oncology indications have been developed, however, only few of them are approved for the treatment of gastrointestinal (GI) cancers, which represent as a huge unmet medical need. As such, we developed a novel ADC of which monomethyl auristatin E (MMAE), an antimitotic agent, was conjugated to a cadherin-17 (CDH17)-targeting monoclonal antibody via protease-cleavable peptide valine-citrulline linker. CDH17 is highly restricted to the intestinal adherent junctions which is not accessible in healthy individuals. In contrast, it is overexpressed and becomes reachable in over half of the gastric and pancreatic cancer patients and in more than 95% of patients with colorectal cancer, making it a perfect target for ADC. Our ADC is designed to deliver MMAE specifically to CDH17-expressing tumors to exert the cytotoxic effect of MMAE on cancer cell, mitigating the undesired off-target cytotoxicity. LC/MS analysis revealed that MMAE was conjugated in a drug-to-antibody ratio of 4 with a yield of higher than 75%. ELISA showed that the binding of naked antibody and ADC to CDH17 was comparable (EC50, 0.17 nM & 0.3 nM, respectively), indicating the conjugation process did not affect antigen binding. This ADC also bound to different CDH17-expressing cell lines, including pancreatic AsPC1 and gastric AGS (EC50, 2.1 nM & 1.53 nM, respectively). By labelling the ADC with pH-sensitive fluorescent indicator, we illustrated that ADC internalization only occurred in CDH17+ but not CDH17- cells, suggesting the high specificity of the ADC. Similarly, the ADC showed selective killing on CDH17+ cells (EC50, 0.02-0.5 nM), and the data together proposed a positive correlation between ADC internalization level and its cytotoxicity (R2 = 0.4307). This supported the specificity and safety of the ADC in killing CDH17+ cancer cells only without affecting normal cells. Furthermore, a preliminary in-vivo study on subcutaneous AsPC1 xenograft model demonstrated that the ADC significantly suppressed tumor growth without causing any changes in body weight. Our ADC would potentially be a “First-in-class” ADC for the treatment of GI cancers with minimal adverse systemic toxicity. Further pharmacokinetics, safety, and other IND-enabling studies are underway. Citation Format: Po Yee Wong, Lin He, Kronos Chow, Kwan Wa Wong, Chui Yee O, Dennis Wong, John Moon Luk, Pui-Chi Lo, Kwong Fai Wong. Novel CDH17-targeting antibody-drug conjugate exhibits anti-tumor efficacy in preclinical models of gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3131.

Abstract 3149: Development of a novel site-specific ADC glycan platform with potential for improved in vivo efficacy and stability of the ADC in animal studies

Abstract Antibody-drug conjugates (ADCs) represent a promising modality for delivering cytotoxic drugs to targeted tumor cells while avoiding off-tumor toxicities. Despite successes in the past 20 years, development of effective ADCs with broad therapeutic window remains challenging due to the complexity of conjugation technologies and the instability of the linkers. Most platforms currently in the market as well as under clinical development may face limitations ascribed to the heterogeneity of ADCs with various drug-to-antibody ratio (DAR). This likely resulted from random conjugation and poor overall biophysical characteristics caused by linker hydrophobicity. The limitations may impact the efficacy, safety, bioavailability, and the robustness of manufacturing process of these therapeutic agents. OBI developed a unique glycan ADC platform (GlycOBI) to enable a site-specific conjugation, which is in a 'Plug and Play' format and compatible with any antibodies, linkers, and payloads in various DAR. This is an efficient and scalable process to generate homogenous ADCs. This platform overcomes the limitations of traditional ADCs, resulting in the improvement of efficacy and stability. We developed a non-genetic, engineering-free approach to generate site-specific homogenous ADCs. This was achieved by utilizing OBI proprietary enzymatic technology (EndoSymeOBI), followed by the click chemistry to conjugate the hydrophilic linker-payload via the glycan site that naturally occurs on the antibody’s Fc region. The conjugation process avoids disrupting the antibody structure, ensuring the related ADC has similar biophysical characteristics compared to native antibody. Furthermore, OBI linker technology improves the conjugation efficiency of the payload, as well as reduces the aggregation propensity, and expands the half-life of the ADC products. Anti-TROP2 and anti-HER2 ADCs are developed based on OBI glycan ADC platform (GlycOBI) using a topoisomerase I inhibitor, exatecan, as the payload. Both ADCs demonstrated greater chemical stability in serum stability studies compared to competitor ADCs. Additionally, they showed broad activity against different cancer cell lines in vitro as well as in mouse xenograft models. The preclinical data presented here suggest that GlycOBI is an outstanding ADC platform to improve the performance of ADCs. Further investigations, including studies in the clinical settings, are warranted. Citation Format: Teng-Yi Huang, Yin-Cheng Hsieh, Ka-Shu Fung, Yu-Chao Huang, Chi-Sheng Shia, Ming-Feng Chiang, Nan-Hsuan Wang, Wan-Fen Li, Ming-Tain Lai. Development of a novel site-specific ADC glycan platform with potential for improved in vivo efficacy and stability of the ADC in animal studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3149.

In vitro and in vivo assessment of gallic acid-chitosan/polycaprolactone conjugate electrospun nanofibers for wound healing

The development of innovative multifunctional wound healing materials based on natural bioactives has attracted a great deal of attention in recent years due to their ability to potentiate the healing process and minimize the challenges linked with conventional wound dressings. The current study demonstrates the development and evaluation of a novel nanofibrous wound healing mat made of Gallic acid-chitosan conjugate (GAC) incorporated within a polycaprolactone (PCL) matrix using an electrospinning technique. The GAC conjugate was synthesized by green synthesis using a free radical polymerization reaction to improve the chitosan's free radical scavenging properties. FTIR, DSC, XRD, and UV spectral analysis characterized the formation of GAC conjugate. The red shift observed in the UV spectrum suggested alterations in electronic transitions resulting from the creation of covalent bonds between Chitosan (CA) and Gallic acid (GA). The appearance of broader and weaker peaks in DSC and the reduced crystallinity in XRD spectrum, signify structural modifications, thereby providing valuable insights into the interaction and structural changes occurring in the GAC conjugates.The SEM image revealed a porous flake-like structural network of GAC conjugate due to the reduced intermolecular hydrogen bonding between them. The functional layer of the nanofibrous mat was produced by electrospinning GAC conjugate with polycaprolactone (PCL) to provide mechanical integrity to the nanofibrous mat. The electrospun nanofibers were characterized using FTIR, DSC, and SEM and evaluated for cell cytotoxicity, angiogenesis, and in vivo wound healing potential in female Wistar rats. The FTIR spectra confirmed the presence of GAC conjugate in the nanofibers, validating the conjugation process. The results demonstrated the successful incorporation of GAC conjugate within the PCL nanofibrous mat, thereby confirming the improvement in the biological properties of the mats. The nanofibrous mat showed angiogenesis activity in the yolk sac membrane assay confirming the property of nanofibers to facilitate cellular adhesion, migration and promotion of angiogenesis with no cytotoxic effect on the viability of mouse stromal cells. On the sixteenth day of therapy, wounds dressed with GAC/PCL nanofibrous mat exhibited a significant improvement in the percent wound closure (94.43%) as compared to the control group (p < 0.05). These results supported the application of GAC/PCL as a novel biomaterial for wound dressing to accelerate the process of wound healing.

Drug deconjugation-assisted peptide mapping by LC–MS/MS to identify conjugation sites and quantify site occupancy for antibody-drug conjugates

Antibody-drug conjugates (ADCs) are a heterogeneous mixture of conjugated species with varied drug loadings. Depending on conjugation sites, linkers and drugs can exhibit different stability as influenced by the solvent-accessibility and local charge, resulting in different ADC efficacy, pharmacokinetics, and toxicity. Conjugation site analysis is critical for ADC structural characterization to assure product quality and consistency. It enables early conjugation studies at site-specific levels, confirms the absence of unexpected products to support conjugation process development, and aids in ensuring lot-to-lot consistency for comparability studies. Peptide mapping using liquid chromatography-tandem mass spectrometry is the industry standard method for analyzing conjugation sites. However, some concerns remain for this approach as the large and hydrophobic drug moieties often result in poor MS/MS fragmentation quality and impede the identification of conjugation sites. Additionally, the ionization discrepancy between conjugated and unconjugated peptides can lead to a relatively large bias for site occupancy calculation. In this work, we present a simple drug deconjugation-assisted peptide mapping method to identify and quantify the drug conjugation for ADCs with protease-cleavable linkers. Papain-based drug deconjugation was used to remove the highly hydrophobic drug moiety, which significantly improved the quantitation accuracy of conjugation level and the fragmentation quality. Sample preparation conditions were optimized to avoid introducing artificial modifications, allowing the tracking of initial sample status and subsequent changes of quality attributes during process development and stability assessment. This method was applied to analyze thermally-stressed ADC samples to monitor changes of site-specific conjugation levels, DAR, succinimide hydrolysis of the linker, and various PTMs. We believe this is an effective and straightforward tool for conjugation site analysis while simultaneously monitoring multiple quality attributes for ADCs with protease-cleavable linkers.

Toward Enhanced Antioxidant and Protective Potential: Conjugation of Corn Cob Xylan with Gallic Acid as a Novel Approach.

Maize ranks as the second most widely produced crop globally, yielding approximately 1.2 billion tons, with corn cob being its primary byproduct, constituting 18 kg per 100 kg of corn. Agricultural corn production generates bioactive polysaccharide-rich byproducts, including xylan (Xyl). In this study, we used the redox method to modify corn cob xylan with gallic acid, aiming to enhance its antioxidant and protective capacity against oxidative stress. The conjugation process resulted in a new molecule termed conjugated xylan-gallic acid (Xyl-GA), exhibiting notable improvements in various antioxidant parameters, including total antioxidant capacity (1.4-fold increase), reducing power (1.2-fold increase), hydroxyl radical scavenging (1.6-fold increase), and cupric chelation (27.5-fold increase) when compared with unmodified Xyl. At a concentration of 1 mg/mL, Xyl-GA demonstrated no cytotoxicity, significantly increased fibroblast cell viability (approximately 80%), and effectively mitigated intracellular ROS levels (reduced by 100%) following oxidative damage induced by H2O2. Furthermore, Xyl-GA exhibited non-toxicity toward zebrafish embryos, offered protection against H2O2-induced stress, and reduced the rate of cells undergoing apoptosis resulting from H2O2 exposure. In conclusion, our findings suggest that Xyl-GA possesses potential therapeutic value in addressing oxidative stress-related disturbances. Further investigations are warranted to elucidate the molecular structure of this novel compound and establish correlations with its pharmacological activities.

Antibody-Drug Conjugate Overview: a State-of-the-art Manufacturing Process and Control Strategy.

Antibody-drug conjugates (ADCs) comprise an antibody, linker, and drug, which direct their highly potent small molecule drugs to target tumor cells via specific binding between the antibody and surface antigens. The antibody, linker, and drug should be properly designed or selected to achieve the desired efficacy while minimizing off-target toxicity. With a unique and complex structure, there is inherent heterogeneity introduced by product-related variations and the manufacturing process. Here this review primarily covers recent key advances in ADC history, clinical development status, molecule design, manufacturing processes, and quality control.The manufacturing process, especially the conjugation process, should be carefully developed, characterized, validated, and controlled throughout its lifecycle. Quality control is another key element to ensure product quality and patient safety. A patient-centric strategy has been well recognized and adopted by the pharmaceutical industry for therapeutic proteins, and has been successfully implemented for ADCs as well, to ensure that ADC products maintain their quality until the end of their shelf life. Deep product understanding and process knowledge defines attribute testing strategies (ATS). Quality by design (QbD) is a powerful approach for process and product development, and for defining an overall control strategy. Finally, we summarize the current challenges on ADC development and provide some perspectives that may help to give related directions and trigger more cross-functional research to surmount those challenges.

Sour cherry seed proteins devoted to covalently bonded carbohydrate moieties: efficacy of transaction and carbohydrate type on amino acid distribution and emulsifier behavior

This paper handled the Maillard reaction under controlled conditions to conjugate sour cherry seed protein isolate (SCPI) with gum Arabic (GA), carboxymethyl cellulose (CMC), and pectin (P) polysaccharides. Characteristic investigations for conjugates were conducted by browning intensity, glycation degree, amino acid, FTIR, SEM, and TGA analyses. After glycation, the level of lysine and arginine declined. Conjugation process displayed beneficial efficacy on techno-functional attributes, namely solubility, water holding capacity, oil holding capacity, foaming capacity/stability and emulsion activity/stability of SCPI but maximum values in terms of these attributes were variable according to conjugate type. Stability index of emulsions stabilized by using conjugates including SCPI-GA (0.87 R), SCPI-CMC (0.94 R), and SCPI-P (0.95 R) was superior than that of SCPI alone (0.60 R). Centrifugal precipitation rate was 59.17, 36.04, 32.61, and 30.92% for emulsions prepared in the presence of SCPI, SCPI-GA, SCPI-CMC, and SCPI-P, respectively. Resilience to freeze-thawed, pH-shifting, various salt concentrations, and different temperature applications of emulsions were investigated to evaluate emulsifier behavior of protein and conjugates in food systems. Findings showed that emulsifying properties of the conjugates prepared using P and CMC came to the fore compared to SCPI alone and SCPI-GA.

Development of Neuroprotective Agents for the Treatment of Alzheimer's Disease using Conjugates of Serotonin with Sesquiterpene Lactones.

Sesquiterpene lactones are secondary plant metabolites with a wide variety of biological activities. The process of lactone conjugation to other pharmacophores can increase the efficacy and specificity of the conjugated agent effect on molecular targets in various diseases, including brain pathologies. Derivatives of biogenic indoles, including neurotransmitter serotonin, are of considerable interest as potential pharmacophores. Most of these compounds have neurotropic activity and, therefore, can be used in the synthesis of new drugs with neuroprotective properties. The aim of this experimental synthesis was to generate potential treatment agents for Alzheimer's disease using serotonin conjugated with natural sesquiterpene lactones. Three novel compounds were obtained via the Michael reaction and used for biological testing. The obtained conjugates demonstrated complex neuroprotective activities. Serotonin conjugated to isoalantolactone exhibited strong antioxidant and mitoprotective activities. The agent was also found to inhibit β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), prevent the aggregation of β-amyloid peptide 1-42, and protect SH-SY5Y neuroblastoma cells from neurotoxins such as glutamate and H2O2. In a transgenic animal model of Alzheimer's disease (5xFAD line), the conjugated agent restored declined cognitive functions and improved learning and memory. In conclusion, the obtained results indicate that serotonin conjugates to sesquiterpene lactones are promising agents for the treatment of symptoms associated with Alzheimer's disease.

Structural and functional characterization of mung bean protein-peach gum conjugate through the Maillard reaction as a novel encapsulation agent

This study used peach gum (PG) and mung bean protein isolate (MBPI) as an encapsulation agent. This study used peach gum (PG) and mung bean protein isolate (MBPI) as an encapsulation agent. The present investigation was undertaken to examine and refine the conjugation process of MBPI-PG, while concurrently evaluating the impact of Maillard modification on functional properties through the wet heat method. Through the analyses of SDS-PAGE, glycation degree, SEM micrographs, and FT-IR spectroscopy, it was ascertained that optimal glycosylation conditions were achieved at a pH of 8.5 and a temperature of 90 °C for a duration of 1 h. Conjugation resulted in the enhanced of solubility, emulsifying properties, and DPPH radical scavenging activity of MBPI. In comparison to their un-conjugated one, the observed improvements in thermal stability and solubility of proteins were attributed to the process of glycation. Collectively, the findings imply that the conjugation with PG imparts additional functional attributes to MBPI, thereby potentially enhancing its applicability in the food industries as a novel encapsulation agent.

Low temperatures do not impair the bacterial plasmid conjugation on poultry meat.

Conjugation plays an important role in the dissemination of antimicrobial resistance genes. Besides, this process is influenced by many biotic and abiotic factors, especially temperature. This study aimed to investigate the effect of different conditions of temperature and storage (time and recipient) of poultry meat, intended for the final consumer, affect the plasmid transfer between pathogenic (harboring the IncB/O-plasmid) and non-pathogenic Escherichia coli organisms. The determination of minimal inhibitory concentrations (MIC) of ampicillin, cephalexin, cefotaxime, and ceftazidime was performed before and after the conjugation assay. It was possible to recover transconjugants in the poultry meat at all the treatments, also these bacteria showed a significant increase of the MIC for all antimicrobials tested. Our results show that a non-pathogenic E. coli can acquire an IncB/O-plasmid through a conjugation process in poultry meat, even stored at low temperatures. Once acquired, the resistance genes endanger public health especially when it is about critically and highly important antimicrobials to human medicine.

Structural insights into the disulfide isomerase and chaperone activity of TrbB of the F plasmid type IV secretion system

Bacteria have evolved elaborate mechanisms to thrive in stressful environments. F-like plasmids in gram-negative bacteria encode for a multi-protein Type IV Secretion System (T4SSF) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SSF assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that the deletion of the disordered N-terminus of TrbBWT, resulting in a truncation construct TrbB37-161, does not affect its catalytic in vitro activity compared to the wild-type protein (p = 0.76). Residues W37–K161, which include the active thioredoxin motif, are sufficient for DI activity. The N-terminus of TrbBWT is disordered as indicated by a structural model of GST-TrbBWT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data and 1H–15N Heteronuclear Single Quantum Correlation (HSQC) spectroscopy of the untagged protein. This disordered region likely contributes to the protein's dynamicity; removal of this region results in a more stable protein based on 1H–15N HSQC and Circular Dichroism Spectroscopies. Lastly, size exclusion chromatography analysis of TrbBWT in the presence of TraW, a T4SSF assembly protein predicted to interact with TrbBWT, does not support the inference of a stable complex forming in vitro. This work advances our understanding of TrbB's structure and function, explores the role of structural disorder in protein dynamics in the context of a T4SSF accessory protein, and highlights the importance of redox-assisted protein folding in the T4SSF.

Study of uricase-polynorbornene conjugates derived from grafting-from ring-opening metathesis polymerization.

PEGylation has been the 'gold standard' in bioconjugation due to its ability to improve the pharmacokinetics and pharmacodynamics of native proteins. However, growing clinical evidence of hypersensitivity reactions to PEG due to pre-existing anti-PEG antibodies in healthy humans have raised concerns. Advancements in controlled polymerization techniques and conjugation chemistries have paved the way for the development of protein-polymer conjugates that can circumvent these adverse reactions while retaining the benefits of such modifications. Herein, we show the development of polynorbornene based bioconjugates of therapeutically relevant urate oxidase (UO) enzymes used in the treatment of gout synthesized by grafting-from ring-opening metathesis polymerization (ROMP). Notably, these conjugates exhibit comparable levels of bioactivity to PEGylated UO and demonstrate increased stability across varying temperatures and pH conditions. Immune recognition of conjugates by anti-UO antibodies reveal low protein immunogenicity following the conjugation process. Additionally, UO conjugates employing zwitterionic polynorbornene successfully avoid recognition by anti-PEG antibodies, further highlighting a potential replacement for PEG.

Influence of the use of psychotropic drugs on the increase of Escherichia coli resistance

Introduction: Escherichia coli is one of the most investigated bacteria due to the diversity of impacts on human health; it can sometimes form new resistant strains through conjugation processes, becoming a real public health problem. Objective: to evaluate the relationship between prolonged use of psychotropic drugs and increased antimicrobial resistance in Escherichia coli strains. Methods: a comprehensive literature review was conducted using different databases of scientific importance; including 35 theses, research articles and reviews published in Spanish and English, from 2019 to 2023. Results: studies of patients under psychotropic treatment with tricyclic antidepressants and selective serotonin reuptake inhibitors for more than 3 months were analyzed; proving the appearance of dysbiosis in the intestinal microbiota. Conclusion: the appearance of dysbiosis in the intestinal microbiota of patients under treatment with psychotropic drugs such as tricyclic antidepressants and selective serotonin reuptake inhibitors, causes a reduction in beneficial bacteria and favors the necessary environment for an increase in pathogenic gram-negative bacteria with resistant mutated strains, specifically Escherichia coli

Kinetic models towards an enhanced understanding of diverse ADC conjugation reactions.

The conjugation reaction is the central step in the manufacturing process of antibody-drug conjugates (ADCs). This reaction generates a heterogeneous and complex mixture of differently conjugated sub-species depending on the chosen conjugation chemistry. The parametrization of the conjugation reaction through mechanistic kinetic models offers a chance to enhance valuable reaction knowledge and ensure process robustness. This study introduces a versatile modeling framework for the conjugation reaction of cysteine-conjugated ADC modalities-site-specific and interchain disulfide conjugation. Various conjugation kinetics involving different maleimide-functionalized payloads were performed, while controlled gradual payload feeding was employed to decelerate the conjugation, facilitating a more detailed investigation of the reaction mechanism. The kinetic data were analyzed with a reducing reversed phase (RP) chromatography method, that can readily be implemented for the accurate characterization of ADCs with diverse drug-to-antibody ratios, providing the conjugation trajectories of the single chains of the monoclonal antibody (mAb). Possible kinetic models for the conjugation mechanism were then developed and selected based on multiple criteria. When calibrating the established model to kinetics involving different payloads, conjugation rates were determined to be payload-specific. Further conclusions regarding the kinetic comparability across the two modalities could also be derived. One calibrated model was used for an exemplary in silico screening of the initial concentrations offering valuable insights for profound understanding of the conjugation process in ADC development.

Influence of selected non-antibiotic pharmaceuticals on antibiotic resistance gene transfer in Escherichia coli.

Antibiotic resistance genes (ARGs) transfer rapidly among bacterial species all over the world contributing to the aggravation of antibiotic resistance crisis. Antibiotics at sub-inhibitory concentration induce horizontal gene transfer (HRT) between bacteria, especially through conjugation. The role of common non-antibiotic pharmaceuticals in the market in disseminating antibiotic resistance is not well studied. In this work, we indicated the effect of some commonly used non-antibiotic pharmaceuticals including antiemetic (metoclopramide HCl) and antispasmodics (hyoscine butyl bromide and tiemonium methyl sulfate) on the plasmid-mediated conjugal transfer of antibiotic resistance genes between pathogenic E. coli in the gastric intestinal tract (GIT). Broth microdilution assay was used to test the antibacterial activity of the tested non-antibiotic pharmaceuticals. A conjugation mating system was applied in presence of the studied non-antibiotic pharmaceuticals to test their effect on conjugal transfer frequency. Plasmid extraction and PCR were performed to confirm the conjugation process. Transmission electron microscopy (TEM) was used for imaging the effect of non-antibiotic pharmaceuticals on bacterial cells. No antibacterial activity was reported for the used non-antibiotic pharmaceuticals. Plasmid-mediated conjugal transfer between isolates was induced by metoclopramide HCl but suppressed by hyoscine butyl bromide. Tiemonium methylsulfate slightly promoted conjugal transfer. Aggregation between cells and periplasmic bridges was clear in the case of metoclopramide HCl while in presence of hyoscine butyl bromide little affinity was observed. This study indicates the contribution of non-antibiotic pharmaceuticals to the dissemination and evolution of antibiotic resistance at the community level. Metoclopramide HCl showed an important role in the spread of antibiotic resistance.

Infusions and Their Pathogenic Significance

This paper explores various disciplines within zoology, emphasizing the interconnectedness of zoology with other biological sciences. Focusing on the evolution of unicellular animals, it investigates the origins of Sporales and Khivchins, with a specific emphasis on infusoria, including their reproductive strategies. The study highlights conjugation and autogamy processes, offering insights into the diversity and evolution of these organisms.