Search Results (113)

Background: Tetanus toxin, produced by Clostridium tetani, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD₅₀) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD₅₀ TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD₅₀ TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs. Full article

Public health crises triggered by viral infections pose severe threats to individual health and disrupt global socioeconomic systems. Against the backdrop of global pandemics caused by highly infectious diseases such as COVID-19 and Ebola virus disease (EVD), the development of innovative prevention and treatment strategies has become a strategic priority in the field of biomedicine. Neutralizing antibodies, as biological agents, are increasingly recognized for their potential in infectious disease control. Among these, nanobodies (Nbs) derived from camelid heavy-chain antibodies exhibit remarkable technical advantages due to their unique structural features. Compared to traditional neutralizing antibodies, nanobodies offer significant cost-effectiveness in production and enable versatile administration routes (e.g., subcutaneous injection, oral delivery, or aerosol inhalation), making them particularly suitable for respiratory infection control and resource-limited settings. Furthermore, engineered modification strategies—including multivalent constructs, multi-epitope recognition designs, and fragment crystallizable (Fc) domain fusion—effectively enhance their neutralizing activity and suppress viral immune escape mechanisms. Breakthroughs have been achieved in combating pathogens such as the Ebola virus and SARS-CoV-2, with mechanisms involving the blockade of virus–host interactions, induction of viral particle disintegration, and enhancement of immune responses. This review comprehensively discusses the structural characteristics, high-throughput screening technologies, and engineering strategies of nanobodies, providing theoretical foundations for the development of novel antiviral therapeutics. These advances hold strategic significance for addressing emerging and re-emerging infectious diseases. Full article

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry. Full article

Background: N-glycosylation is a post-translational modification involving the attachment of oligosaccharides to proteins and is known to influence immunoglobulin G (IgG) effector functions and even antigen binding. IgG contains an evolutionarily conserved N-glycosylation site in its fragment crystallizable (Fc) region, while during V-D-J recombination and somatic hypermutation processes it can also obtain N-glycosylation sites in its antigen binding fragment (Fab). Our previous study demonstrated altered IgG N-glycosylation in children at type 1 diabetes (T1D) onset, with the most prominent changes involving sialylated glycans, hypothesized to mainly come from the Fab region, however, the analytical method used could not distinguish between Fc and Fab. Methods: IgG was isolated from plasma from 118 children with T1D and 98 healthy controls from the Danish Registry of Childhood and Adolescent Diabetes. Isolated IgG was cleaved into Fc and Fab fragments using IdeS enzyme. N-glycans were enzymatically released from each fragment, fluorescently labelled with procainamide, and analyzed separately using the UPLC-MS method. Structural annotation of resulting chromatograms was performed using MS/MS. Results: T1D related N-glycosylation changes were more pronounced in the Fab glycans compared to Fc glycans, with five Fab glycans (Man5, Man7, FA2BG1S1, A2G2S2, FA2BG2S1) being significantly altered compared to only one in the Fc region (FA2[3]BG1). Comparing Fc and Fab glycosylation overall reveals stark differences in the types of glycans on each region, with a more diverse and complex repertoire being present in the Fab region. Conclusions: These findings suggest that N-glycosylation changes in early onset T1D predominantly originate from the Fab region, underscoring their potential role in modulating (auto)immunity and highlighting distinct glycosylation patterns between Fc and Fab. Full article

To understand the effect of the hydroxyl group and processing temperatures on dielectric losses of flame retardants and flame-retardant polymers, the performance difference between 6-methyldibenzo[c,e][1,2]oxaphosphinine 6-oxide (DOPO-Me) and 6-(hydroxymethyl)dibenzo[c,e][1,2]oxaphosphinine 6-oxide (DOPO-HM) has been investigated, respectively, in non-polar and polar polymers at 7–20 GHz. DOPO-HM and DOPO-Me differ by only one OH group. The former demonstrates a lower dissipation factor (Df) than the latter, owing to hydrogen bonds. In polystyrene and crosslinked polyphenylene oxide, both flame retardants increase a dielectric loss of flame-retardant polymers, with DOPO-HM being less detrimental because of its higher crystallizability and lower plasticization. In polar poly(methyl methacrylate) (PMMA), conformational changes in PMMA main chains caused by flame retardants and high processing temperatures lead to an early Df drop of PMMA at low loadings of the flame retardants. At high loadings, a change in the physical form of flame retardants from a primitive crystalline state to an amorphous state increases a dielectric loss of flame retardant PMMA, with DOPO-HM resulting in a slightly higher dielectric loss than DOPO-Me. These results prove that the effect of a hydroxyl group in organophosphorus structures on the dielectric loss of flame-retardant polymers is crucially dependent on its interaction with the polymer matrix. Full article

A newly developed series of polylactide (PLA)-based composites filled with hybrid lignin–carbon nanotube (CNTs) particles were studied using thermal and dielectric techniques. The low CNT content (up to 3 wt%) aimed to create conductive networks while enhancing particle–polymer adhesion. For comparison, PLA composites based on lignin and CNTs were also examined. Although infrared spectroscopy showed no significant interactions, calorimetry and dielectric spectroscopy revealed a rigid interfacial PLA layer exhibiting restricted mobility. The interfacial polymer amount was found to increase monotonically with the particle content. The hybrid-filled PLA composites exhibited electrical conductivity, whereas PLA/Lignin and PLA/CNTs remained insulators. The result was indicative of a synergistic effect between lignin and CNTs, leading to lowering of the percolation threshold to 3 wt%, being almost ideal for sustainable conductive printing inks. Despite the addition of lignin and CNTs at different loadings, the glass transition temperature of PLA (60 °C) decreased slightly (softer composites) by 1–2 K in the composites, while the melting temperature remained stable at ~175 °C, favoring efficient processing. Regarding crystallization, which is typically slow in PLA, the hybrid lignin/CNT particles promoted crystal nucleation without increasing the total crystallizable fraction. Overall, these findings highlight the potential of eco-friendly conductive PLA composites for new-generation applications, such as printed electronics. Full article

Allostery is a fundamental biological phenomenon that occurs when a molecule binds to a protein’s allosteric site, triggering conformational changes that regulate the protein’s activity. However, allostery in antibodies remains largely unexplored, and only a few reports have focused on allostery from antigen-binding fragments (Fab) to crystallizable fragments (Fc). But this study, using anti-phenobarbital antibodies—which are widely applied for detecting the potential health food adulterant phenobarbital—as a model and employing multiple computational methods, is the first to identify a cyclopeptide (cyclo[Link-M-WFRHY-K]) that induces allostery from Fc to Fab in antibody and elucidates the underlying antibody allostery mechanism. The combination of molecular docking and multiple allosteric site prediction algorithms in these methods identified that the cyclopeptide binds to the interface of heavy chain region-1 (CH₁) in antibody Fab and heavy chain region-2 (CH₂) in antibody Fc. Meanwhile, molecular dynamics simulations combined with other analytical methods demonstrated that cyclopeptide induces global conformational shifts in the antibody, which ultimately alter the Fab domain and enhance its antigen-binding activity from Fc to Fab. This result will enable cyclopeptides as a potential Fab-targeted allosteric modulator to provide a new strategy for the regulation of antigen-binding activity and contribute to the construction of novel immunoassays for food safety and other applications using allosteric antibodies as the core technology. Furthermore, graph theory analysis further revealed a common allosteric signaling pathway within the antibody, involving residues Q123, S207, S326, C455, A558, Q778, D838, R975, R1102, P1146, V1200, and K1286, which will be very important for the engineering design of the anti-phenobarbital antibodies and other highly homologous antibodies. Finally, the non-covalent interaction analysis showed that allostery from Fc to Fab primarily involves residue signal transduction driven by hydrogen bonds and hydrophobic interactions. Full article

IgG4 is an unusual immunoglobulin (Ig) and is the least component of IgG in humans. It is often asymmetrical and heterobivalent with weak Fc (fragment crystallizable region)-dependent effector function and ineffective complement activation, thus playing an unclear role in immune functions. IgE is an uncommon Ig, being important mostly in allergy and type 2 immunity. There are two rare chronic Ig-related fibroinflammatory diseases, namely IgG4-related disease (IgG4RD) and Kimura disease (KD), characterized by prominent IgG4- or IgE-positive plasma cells in the affected tissues, with or without blood elevations of the same Ig. The etiology of these two Ig-related diseases is unclear, though it appears that the pathogenesis in both is related to polarized Ig heavy chain isotype switching, concomitant with other cellular, cytokine and chemotaxin interactions that culminates in the characteristic pathologic manifestations of inflammation and fibrosis. IgG4RD and KD, despite having overlapping and differing features, may be connected by the similar pathogenetic polarized Ig isotype switching. Full article

This study investigates the biodegradation behavior of poly(butylene succinate) (PBS) copolyesters containing dilinoleic acid (DLA) co-monomeric units and wood flour (WF) as a filler. PBS-DLA is a segmented thermoplastic elastomer (TPE), where the soft amorphous phase is formed by DLA ester segments, while the hard phase consists of crystallizable PBS domains. Wood–plastic composites (WPCs) were prepared with WF at weight fractions of 10%, 20%, 30%, and 40% wt. and analyzed in terms of surface morphology, chemical structure, mechanical performance, and thermal stability before and after biodegradation in soil conditions. The results of microscopic analysis confirmed that the PBS-DLA copolymer and its composites undergo surface biodegradation as manifested by increased surface roughness and microcrack formation, particularly in composites with a higher WF content. ATR FT-IR spectroscopy indicated oxidation and hydrolysis, supporting the hypothesis of progressive surface erosion. Mechanical tests showed a decline in tensile strength and elongation at break, with the most pronounced changes in composites containing 20% WF. Thermal analysis (DSC, DMTA, and TGA) confirmed that the PBS-DLA copolymer retains its thermoplastic elastomeric behavior after a 3-month biodegradation experiment. The storage modulus (E′) remained stable, while only minor variations in melting and crystallization temperatures were observed. These findings reinforce the hypothesis of surface erosion rather than a bulk degradation mechanism. Given their biodegradability and retained thermoplastic behavior, WPC composites based on PBS-DLA copolyester could be promising for eco-friendly applications where controlled degradation is desirable, such as in packaging, agriculture, or biodegradable consumer goods. Full article

Protein crystallization is an alternative to well-established but cost-intensive and time-consuming chromatography in biotechnological processes, with protein crystallization defined as an essential unit operation for isolating proteins, e.g., active pharmaceutical ingredients. Crystalline therapeutic proteins attract interest in formulation and delivery processes of biopharmaceuticals due to the high purity, concentration, and stability of the crystalline state. Although improving protein crystallization is mainly achieved by high-throughput screening of crystallization conditions, recent studies have established a rational protein engineering approach to enhance crystallization for two homologous alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefiri (LkADH). As generalizing crystallization processes across a wide range of target proteins remains challenging, this study takes a further step by applying the successful crystal contact engineering strategies for LbADH/LkADH to a non-homologous protein, an NADH-binding derivative of the Nostoc sp. PCC 1720 ene reductase (NspER1-L1,5). Here, the focus lies on introducing electrostatic interactions at crystal contacts, specifically between lysine and glutamic acid. Out of the nine tested NspER1-L1,5 mutants produced in E. coli, six crystallized, while four mutants revealed an increased propensity to crystallize in static µL-batch crystallization compared to the wild type: Q204K, Q350K, D352K, and T354K. The best-performing mutant Q204K was selected for upscaling, crystallizing faster than the wild type in a stirred batch crystallizer. Even when spiked with E. coli cell lysate, the mutant maintained increased crystallizability compared to the wild type. The results of this study highlight the potential of crystal contact engineering as a reliable tool for improving protein crystallization as an alternative to chromatography, paving the way for more efficient biotechnological downstream processing. Full article

A fiber-reinforced SPR sensor based on silver-nucleated gold-shell bimetallic nanoparticles and graphene oxide was developed and applied to human IgG detection. The refractive index (RI) sensitivity of the Ag@Au/GO fiber SPR sensor is as high as 4715.9 nm/RIU in the RI range of 1.333–1.365. Staphylococcus aureus protein A (SPA) can specifically recognize and bind to the fragment crystallizable (Fc) of the antibody; it facilitates the highly targeted immobilization of the antibody. SPA and rabbit anti-human IgG were immobilized on the surface of the Ag@Au/GO fiber SPR sensor for the detection of different concentrations of human IgG with a sensitivity of 0.53 nm/μg/mL and detection limits of 0.037 μg/mL. This biosensor based on the mixed structure of GO and Ag@Au combined the common advantages of the two materials. Therefore, our study provides a simple platform for biological analysis and has a good application prospect. Full article

Background: Monoclonal antibodies (mAbs) are potent treatment options for infectious diseases. The rapid isolation and in vivo validation of therapeutic mAb candidates, including mAb cocktails, are essential to combat novel or rapidly mutating pathogens. The rapid selection and production of mAb candidates in sufficient amount and quality for preclinical studies are a major limiting step in the mAb development pipeline. Methods: Here, we developed a method to facilitate the screening of therapeutic mAbs in mouse models. Four conventional mAbs were transformed into single-chain variable fragments fused to the fragment crystallizable (Fc) region of a human IgG1 (scFv-IgG). These scFv-IgG were expressed individually or as a cocktail in vitro and in mice following transfection or hydrodynamic delivery of the corresponding plasmids. Results: This method induced high expression of all scFv-IgG and provided protection in two murine infection models. Conclusions: This study highlights the benefits of this approach for the rapid, low-cost screening of therapeutic mAb candidates. Full article

Rheumatoid factor (RF) is the first autoantibody identified in rheumatoid arthritis (RA) which targets the fragment crystallizable (Fc) region of immunoglobulin (Ig) G. Although IgM isotype is predominant, other Ig isotypes, including IgG and IgA, also exist. While RF is not specific to RA, it remains a valuable serological test for diagnosing the disease, as evidenced by its inclusion in the 2010 classification criteria for RA based on elevated serum RF levels. RF is also associated with RA severity, including joint damage and extra-articular manifestations, serving as a poor prognostic factor and aiding in the identification of difficult-to-treat RA. Recent studies have demonstrated that high serum RF levels are associated with a reduced response to tumor necrosis factor (TNF) inhibitors. In contrast, anti-TNF antibodies lacking the Fc portion have shown stable efficacy in RA patients regardless of baseline RF levels. These findings reaffirm the clinical significance of RF measurement, 80 years after its initial discovery. This review explores the diagnostic and prognostic significance of RF and its impact on treatment selection in RA management. Full article

The present work aimed to evaluate the extractive performance of three green solvents—absolute ethanol, hydrated ethanol (96%), and absolute isopropanol (AIP)—in high stearic high oleic sunflower seeds, comparing them with the conventional solvent hexane. The oil yield from exhaustive Soxhlet extraction with hydrated ethanol was significantly lower, with no significant differences being observed among the other solvents. Extraction with AIP produced the extract with the lowest non-lipid material content and the oil with the lowest concentration of crystallizable waxes, showing a 53% reduction compared to hexane. Since AIP showed a higher extraction efficiency than absolute ethanol after 4 h of processing, its oil extraction kinetics when used as a solvent were further studied. A modified Fick’s diffusion model revealed that, for hexane extraction at 50 °C, the effective diffusion coefficient and the washing fraction were higher than those for AIP extraction (26% and 5.4% higher, respectively). No clear dependence of the oil extraction kinetics on the temperature was observed between the studied temperatures (50 °C and 70 °C). The results showed the feasibility of using absolute ethanol and AIP as alternatives to hexane. Additionally, isopropanol presented operational advantages, producing oil that required less dewaxing during refining than that extracted with hexane or ethanol and showing higher oil selectivity than ethanol. Full article

A series of poly(lactic acid) (PLA)/biochar (BC) bio-composites filled with low amounts (1–5 wt%) of BC were prepared and characterized. The synthesis involved the in situ ring-opening polymerization (ROP) of lactide in the presence of two different types of BC named SWP550 and SWP700, having been produced by pyrolysis of softwood pellets at two different temperatures, 550 and 700 °C, respectively. The bio-composites were characterized by complementary techniques. The successful synthesis of PLA and PLA/BC bio-composites was directly demonstrated by the formation of new bonds, most probably between PLA and BC. Indirect evidence for that was obtained by the systematic molar mass reduction in the presence of BC. BC was found by transmission electron microscopy (TEM) micrographs to be well dispersed at the nanosize level, indicating that in situ polymerization is a technique quite efficient for producing bio-composites with finely dispersed BC additive. The molecular dynamics mapping is performed here via dielectric spectroscopy, moreover, for the first time in these PLA/BC systems. The strong PLA/BC interactions (due to the grafting) led to a systematic deceleration of segmental mobility (elevation of the T_g) in the bio-composites despite the opposite effect expected by the decrease in molar mass with the BC content increasing. In addition, the same interactions and chain-length reduction are responsible for the slight suppression of the PLA’s crystallizability. The effects are slightly stronger for SWP700 as compared to SWP550. The crystal structure is rather similar between the unfilled matrix and the bio-composites, whereas, based on the overall data, the semicrystalline morphology is expected to be tighter in the bio-composites. The thermal stability and decomposition kinetics were also thoroughly studied. All materials exhibit good resistance to thermal degradation. Additionally, the mechanical properties of bio-composites were evaluated by tensile testing and found slightly enhanced at low biochar contents and decreasing thereafter due to the low molecular weight of bio-composites with the larger BC contents. Full article