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Methods in Molecular Biology (Clifton,... 2022Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and...
Single-domain antibodies (sdAbs) are binders that consist of a single immunoglobulin domain. SdAbs have gained importance as therapeutics, diagnostic reagents, and research tools. Functional sdAbs are commonly produced in Escherichia coli, which is a simple and widely used host for production of recombinant proteins. However, there are drawbacks of the E. coli expression system, including the potential for misfolded recombinant proteins and pyrogenic contamination with toxic lipopolysaccharides. Pichia pastoris is an alternative host for the production of heterologous proteins because of its high recombinant protein yields and the ability to produce soluble, properly folded proteins without lipopolysaccharide contamination. Here, we describe a method to produce sdAbs in P. pastoris. We present methods for the cloning of sdAb-encoding genes into a P. pastoris expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.
Topics: Escherichia coli; Pichia; Recombinant Proteins; Saccharomycetales; Single-Domain Antibodies
PubMed: 35157274
DOI: 10.1007/978-1-0716-2075-5_9 -
Seminars in Dialysis Sep 2022High-volume hemodiafiltration involves filtration of >23 L/treatment and its replacement by sterile non-pyrogenic substitution fluid, while maintaining the patient's...
High-volume hemodiafiltration involves filtration of >23 L/treatment and its replacement by sterile non-pyrogenic substitution fluid, while maintaining the patient's fluid balance. That volume of substitution fluid precludes the use of prepackaged sterile fluid. Instead, substitution fluid must be prepared on-line using machines that incorporate a series of bacteria- and endotoxin-retentive filters. The sterilizing ultrafilters are validated to deliver sterile, non-pyrogenic fluid to the patient when operated according to the machine manufacturer's instructions and in compliance with international standards and regulatory oversight. A successful hemodiafiltration program also places important responsibilities on the user. Specifically, the user is responsible for ensuring that the dialysis water or dialysis fluid delivered to the sterilizing filters of the hemodiafiltration machine meets the machine manufacturer's specifications and is consistent with the quality used in the sterilization validation process. The user is also responsible for ensuring that the treatment prescription allows a filtration volume >23 L/treatment to be achieved by careful selection of a dialyzer, blood flow rate and treatment time. Questions related to assurance that the substitution fluid will routinely be sterile and non-pyrogenic have limited the uptake of on-line hemodiafiltration as a therapeutic option in some countries, such as the United States.
Topics: Dialysis Solutions; Endotoxins; Hemodiafiltration; Humans; Renal Dialysis; Water
PubMed: 35315951
DOI: 10.1111/sdi.13073 -
Advances in Clinical Chemistry 2024The outer membrane of gram-negative bacteria is primarily composed of lipopolysaccharide (LPS). In addition to protection, LPS defines the distinct serogroups used to...
The outer membrane of gram-negative bacteria is primarily composed of lipopolysaccharide (LPS). In addition to protection, LPS defines the distinct serogroups used to identify bacteria specifically. Furthermore, LPS also act as highly potent stimulators of innate immune cells, a phenomenon essential to understanding pathogen invasion in the body. The complex multi-step process of LPS binding to cells involves several binding partners, including LPS binding protein (LBP), CD14 in both membrane-bound and soluble forms, membrane protein MD-2, and toll-like receptor 4 (TLR4). Once these pathways are activated, pro-inflammatory cytokines are eventually expressed. These binding events are also affected by the presence of monomeric or aggregated LPS. Traditional techniques to detect LPS include the rabbit pyrogen test, the monocyte activation test and Limulus-based tests. Modern approaches are based on protein, antibodies or aptamer binding. Recently, novel techniques including electrochemical methods, HPLC, quartz crystal microbalance (QCM), and molecular imprinting have been developed. These approaches often use nanomaterials such as gold nanoparticles, quantum dots, nanotubes, and magnetic nanoparticles. This chapter reviews current developments in endotoxin detection with a focus on modern novel techniques that use various sensing components, ranging from natural biomolecules to synthetic materials. Highly integrated and miniaturized commercial endotoxin detection devices offer a variety of options as the scientific and technologic revolution proceeds.
Topics: Animals; Humans; Rabbits; Endotoxins; Lipopolysaccharides; Gold; Metal Nanoparticles; Cytokines
PubMed: 38280803
DOI: 10.1016/bs.acc.2023.11.001 -
Carbohydrate Polymers Aug 2023Bacterial cellulose (BC) is a chemically pure, non-toxic, and non-pyrogenic natural polymer with high mechanical strength and a complex fibrillar porous structure. Due... (Review)
Review
Bacterial cellulose (BC) is a chemically pure, non-toxic, and non-pyrogenic natural polymer with high mechanical strength and a complex fibrillar porous structure. Due to these unique biological and physical properties, BC has been amply used in the food industry and, to a somewhat lesser extent, in medicine and cosmetology. To expand its application the BC structure can be modified. This review presented some recent developments in electrically conductive BC-based composites. The as-synthesized BC is an excellent dielectric. Conductive polymers, graphene oxide, nanoparticles and other materials are used to provide it with conductive properties. Conductive bacterial cellulose (CBC) is currently investigated in numerous areas including electrically conductive scaffolds for tissue regeneration, implantable and wearable biointerfaces, flexible batteries, sensors, EMI shielding composites. However, there are several issues to be addressed before CBC composites can enter the market, namely, composite mechanical strength reduction, porosity decrease, change in chemical characteristics. Some of them can be addressed both at the stage of synthesis, biologically, or by adding (nano)materials with the required properties to the BC structure. We propose several solutions to meet the challenges and suggest some promising BC applications.
Topics: Cellulose; Polymers; Prostheses and Implants; Electronics; Nanoparticles
PubMed: 37182950
DOI: 10.1016/j.carbpol.2023.120850 -
Biotechnologia 2022Liposomes (LSs) are promising nanoparticles with unique properties such as controlled nanosize, large surface area, increased reactivity, and ability to undergo... (Review)
Review
Liposomes (LSs) are promising nanoparticles with unique properties such as controlled nanosize, large surface area, increased reactivity, and ability to undergo modification. Worldwide, licensed liposomal forms of antibiotics, hormones, antioxidants, cytostatics, ophthalmic drugs, etc., are available on the pharmaceutical market. This review focuses on the adjuvant properties of LSs in the production of vaccines (VACs). LS-VACs have the following advantages: antigens with low immunogenicity can become highly immunogenic; LSs can include both hydrophilic and hydrophobic antigens; LSs allow to achieve a prolonged specific action of antibodies; and LSs reduce the toxicity and pyrogenicity of encapsulated antigens and adjuvants. The immune response is influenced by the composition of the liposomal membrane, physicochemical characteristics of lipids, antigen localization in LSs, interaction of LSs with complement, and a number of proteins, which leads to opsonization. The major requirements for adjuvants are their ability to enhance the immune response, biodegradability, and elimination from the organism, and LSs fully meet these requirements. The effectiveness and safety of LSs as carriers in the antigen delivery system have been proven by the long-term clinical use of licensed vaccines against hepatitis A, influenza, herpes zoster, malaria, and COVID-19.
PubMed: 36685697
DOI: 10.5114/bta.2022.120709 -
European Journal of Medicinal Chemistry May 2024Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl... (Review)
Review
Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl dipeptide (MDP, N-acetyl muramyl L-alanyl-d-isoglutamine), identified as the minimal immunologically active component of bacterial cell wall peptidoglycan (PGN) is recognized by NOD2. In terms of biological activities, MDP demonstrated vaccine adjuvant activity and stimulated non-specific protection against bacterial, viral, and parasitic infections and cancer. However, MDP has certain drawbacks including pyrogenicity, rapid elimination, and lack of oral bioavailability. Several detailed structure-activity relationship (SAR) studies around MDP scaffolds are being carried out to identify better NOD2 ligands. The present review elaborates a comprehensive SAR summarizing structural aspects of MDP derivatives in relation to NOD2 agonistic activity.
Topics: Nod2 Signaling Adaptor Protein; Acetylmuramyl-Alanyl-Isoglutamine; Structure-Activity Relationship; Humans; Animals; Molecular Structure
PubMed: 38691886
DOI: 10.1016/j.ejmech.2024.116439 -
International Dental Journal Jun 2023A significant increase in the incidence of scarlet fever, mainly in Europe, has been noted during the COVID-19 postpandemic period. Scarlet fever is caused by a... (Review)
Review
A significant increase in the incidence of scarlet fever, mainly in Europe, has been noted during the COVID-19 postpandemic period. Scarlet fever is caused by a pyrogenic exotoxin-producing streptococcus-Streptococcus pyogenes-responsible for more than 500,000 deaths annually worldwide. Superantigens (SAgs) secreted by this Group A streptococcus (GAS) usually overstimulate the human immune system, causing an amplified hypersensitivity reaction leading to initial symptoms such as sore throat, high fever, and a sandpaper-like skin rash. There could be concurrent oral manifestations known as "strawberry tongue" or "raspberry tongue," which may be first noted by oral health professionals. The early diagnosis and treatment of this disease is critical to obviate the development of local and systemic sequelae such as acute rheumatic fever, endocarditis, and glomerulonephritis. Antibiotics should be prescribed early to mitigate its duration, sequelae, and community spread. Dental practitioners should be aware of the early symptoms of scarlet fever for infection detection, emergency patient management, and appropriate referral. This concise review outlines the prevalence, pathogenicity, oral and systemic manifestations, as well as the dental implications of scarlet fever.
Topics: Humans; Scarlet Fever; Dentists; COVID-19; Professional Role; Streptococcus pyogenes; Recurrence
PubMed: 37062653
DOI: 10.1016/j.identj.2023.03.009 -
Environmental Science & Technology Sep 2023Quinones and products of their redox reactions (hydroquinones and semiquinones) have been suggested as important players in the reductive dehalogenation of...
Quinones and products of their redox reactions (hydroquinones and semiquinones) have been suggested as important players in the reductive dehalogenation of organohalogens mediated by natural and pyrogenic organic matter, although based on limited direct evidence. This study focused on the reductive dehalogenation of a model organohalogen (triclosan) by 1,4-benzohydroquinone (HQ). In the presence of HQ only, degradation of triclosan does not occur within the experimental period (up to 288 h); however, it takes place in the presence of HQ and FeCl under anoxic conditions at pH 5 and 7 (above the p of SQ = 4.1) only to be halted in the presence of dissolved oxygen. Kinetic simulation and thermodynamic calculations indicated that benzosemiquinone (SQ) is responsible for the reductive degradation of triclosan, with the fitted rate constant for the reaction between SQ and triclosan being 317 M h. The critical role of semiquinones in reductive dehalogenation can be relevant to a wide range of quinones in natural and engineering systems based on the reported oxidation-reduction potentials of quinones/semiquinones and semiquinones/hydroquinones and supported by experiments with additional model hydroquinones.
Topics: Hydroquinones; Triclosan; Computer Simulation; Ketones; Quinones
PubMed: 37668505
DOI: 10.1021/acs.est.3c03981 -
BioRxiv : the Preprint Server For... Apr 2023Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods...
Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [F]FDG, yet lack specificity to the causative pathogen () and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian disaccharide trehalose - 2-[F]fluoro-2-deoxytrehalose ([F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [F]FDT in the imaging of in diverse models of disease, including non-human primates, successfully co-opts -specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [F]FDT from the most globally-abundant organic F-containing molecule, [F]FDG. The full, pre-clinical validation of both production method and [F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [F]FDT directly from the widely-available clinical reagent [F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.
PubMed: 37333343
DOI: 10.1101/2023.04.03.535218 -
Biologicals : Journal of the... Jun 2022To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to...
To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to generate stably transfected cells. Through stimulation with pyrogens, a signal was obtained that was dose-dependent with the concentration of pyrogen. Using the cells, we selected and optimized the parameters and found that the optimal conditions may be with 5 × 10/ml cells that were seeded and incubated with pyrogen for 3-6 h in IMDM medium with 2% FBS. Based on the optimized parameters, a novel RGA was developed. Then, the RGA was validated and the results showed that the linearity was greater than 0.95 between the signals and the concentrations of pyrogen, the recoveries of pyrogen were all between 50% and 200%, and the precision was less than 35%. There was no difference in the sensitivity, specificity or reproducibility between RGA and BET, and the results from RGA and MAT and RPT were consistent. Furthermore, the RGA can be applied to the pyrogen detection of monoclonal antibodies. Due to its advantages including a fast detection speed, high sensitivity, convenient mode of operation and wide-pyrogen spectrum detection, RGA is promising as a supplementary method to detect pyrogen.
Topics: Biological Assay; Genes, Reporter; Luciferases; Pyrogens; Reproducibility of Results
PubMed: 35729037
DOI: 10.1016/j.biologicals.2022.05.003