Escherichia coli is one of the most common producers of recombinant proteins, including therapeutic antibody fragments. However, the outer membrane of E. coli contains high levels of lipopolysaccharide (LPS, also known as endotoxin), which can activate innate immune receptors, trigger immune responses, and induce systemic inflammation that may progress to septic shock. Ensuring extremely low endotoxin levels in preparations intended for in vivo applications is critically important. In this study, we investigated the endotoxin content in preparations of the bispecific mini-antibody MYSTI-2 produced in two E. coli strains: the Rosetta strain, which synthesizes conventional LPS, and the ClearColi strain, which synthesizes potentially non-toxic form of LPS. Our results demonstrate that near-complete removal of LPS can be achieved only through the use of a non-ionic detergent during purification, regardless of the bacterial strain used for protein production.
Gradient hydrogels represent a unique class of biomaterials capable of mimicking the spatial heterogeneity of native tissues and providing targeted effects on cells through mechanical, chemical, and biophysical gradients. In recent years, numerous fabrication strategies have been developed to generate gradient hydrogels, including layer-by-layer formation, photopolymerization, microfluidic techniques, and 3D/4D printing. This review summarizes current methodologies for the characterization of gradient hydrogels and highlights their emerging biomedical applications, such as controlled drug delivery, tissue engineering, regenerative medicine, organ-on-chip systems, and soft bioelectronic devices. Furthermore, the review discusses critical challenges related to the protocol standardization, manufacturing scalability, integration with additive manufacturing technologies, and potential regulatory barriers.
Mitochondria are semi-autonomous, multifunctional organelles that supply cells with energy. They are highly dynamic structures, capable of moving, fusing, dividing, and forming branched networks. The number, density, and complexity of mitochondrial network are unique to each cell type and reflect cellular demands for ATP and other mitochondria-dependent metabolites. Mitochondrial dysfunction is a hallmark of many neurodegenerative diseases; however, the relationships between neurodegeneration and mitochondrial morphogenesis, intracellular localization, and dynamics remain incompletely understood. Interpretation and comparison of published data are complicated by the diversity of analytical approaches used to study mitochondrial behavior. In this research, we investigated the effects of a pathogenic mutation in the huntingtin protein (HTT), which causes Huntington's disease (HD), on mitochondrial morphology and motility, with particular emphasis on associated disruptions in the cytoskeletal organization. We performed a systematic evaluation of automated mitochondrial analysis tools and selected MiNA, TrackMate, and JACoP as the optimal platforms for quantitative assessment of the effects of mutant HTT (mHTT) on the mitochondrial morphology, motility, and interaction with cytoskeletal components and identification of specific disruptions directly related to HD pathogenesis. Our analysis revealed that mitochondria in mHTT-expressing cells are significantly shorter, more branched, and less motile than in control cells. Moreover, their interactions with microtubules and vimentin intermediate filaments are markedly altered. Together, these findings establish a link between HD and specific defects in the mitochondrial network, thus contributing to understanding cellular mechanisms of HD development, and suggest that mHTT disrupts the interaction of mitochondria with cytoskeletal components responsible for their movement and distribution in the cell, thereby negatively affecting mitochondrial motility and morphology.
Cytokines play a critical role in brain functioning by modulating neurotransmitter and energy metabolism, neuroplasticity, and neuronal activity. Dysregulated or excessive cytokine production can disrupt neuronal metabolic processes and contribute to brain dysfunction. Among the proposed mechanisms underlying the development and progression of affective disorders (ADs), the cytokine hypothesis emphasizes the role of inflammatory markers as key factors in the development of depressive pathologies. The aim of this study was to investigate molecular characteristics of selected immunoinflammatory markers in patients with AD. The study included 239 patients diagnosed with AD and 205 healthy controls. Polymorphic variants of the immunoinflammatory genes IL1B (rs16944, rs1143627), IL13 (rs1295686), TNFB (rs2229094), and TGFA (rs2166975) were analyzed, and cytokine levels in the blood serum and peripheral blood mononuclear cells were measured. As association was identified between the rs2229094 polymorphism of the TNFB gene and AD: the carriage of the A allele and the AA genotype of this variant was associated with an increased risk of AD. Furthermore, the levels of TGF-α and IL-13 in peripheral blood mononuclear cells and the serum content of TNF-β were significantly elevated in patients with AD compared with healthy controls. These pilot findings suggest that the studied cytokines may contribute to the pathogenetic mechanisms underlying development of ADs.
Modern magnetic resonance imaging (MRI) methods enable individualized assessment of both functional brain activity and neurochemical composition. Functional magnetic resonance imaging (fMRI) allows evaluation of brain activity at rest and during task performance, while magnetic resonance spectroscopy (MRS) provides measurements of key metabolites such as choline, N-acetylaspartate, creatine, lactate, lipids, alanine, glutamine and glutamate, GABA, and myo-inositol. These approaches are widely used in both fundamental brain research and diagnostic studies. However, existing literature lacks methods for directly comparing these individual assessments, which is essential for investigating relationships between metabolite levels and brain activity. Here, we present a method for aligning individual fMRI and MRS data. Using this approach, we demonstrated a neurophysiological phenomenon in which the functional connectivity between brain regions increases while overall functional activity decreases during task performance.
Widespread interest and broad application of alginic acid and its salts in tissue engineering, regenerative medicine, biotechnology, and pharmaceutical industry is due to the several unique properties: biomechanical compatibility with living tissue, lack of toxicity, and bioabsorption capacity. This literature review analyzes effects of the anionic structure of the binary alginate copolymer on physicochemical properties of the resulting solutions and gels, as well as characteristics and conditions of their processing to obtain functional products for medical and biological applications. Dependence of functionality of the products on the ratio of M(β-D-mannuronate)/G(α-L-guluronate) blocks in the chain and on the source of alginate are also considered. Influence of the quantitative content of guluronic (G) acid blocks in the chain of linear alginate on its susceptibility to H+-induced gelation is described. A review of the mechanisms of gelling in the alginate solutions caused by formation of ionic, hydrogen, and covalent bonds is provided. In particular, attention is paid to the rate of dissolution of alginate salts, viscosity properties of the solutions based on them, and their dependence on ionic strength and pH. The mechanisms of interaction between both native and chemically modified alginates with various biologically active substances, drugs, and surfactants are considered. A detailed study of these processes opens new possibilities not only for obtaining dimensionally stable gels for tissue engineering structures, but also for obtaining systems designed for the controlled release of drugs.
DNA-dependent nuclear enzymes poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) are involved in the regulation of multiple DNA repair pathways, including base excision repair (BER). After activation by binding to damaged DNA, these enzymes synthesize negatively charged poly(ADP-ribose) (PAR) and covalently attach to amino acid residues of target proteins, including PARPs themselves. PARP2 activity is influenced by the nature of DNA lesion; for example, it is efficiently stimulated by DNA breaks flanked by phosphate group. However, it remains unclear which stages of the auto-PARylation reaction are most sensitive to the structure of damaged DNA. In this study, we investigated how PARP2 activity depends on the presence and position of a single-nucleotide gap in DNA (either free or in the context of nucleosome) at different stages of the automodification reaction conducted in the absence of the histone PARylation factor HPF1. The obtained results suggest that the presence of the gap affects the affinity of PARP2 for DNA/nucleosomes, thereby determining the number of catalytically active enzyme molecules and the efficiency of PARylation initiation. In contrast, PAR elongation was affected by the lesion location in the DNA/nucleosome structure, namely, its distance from the blunt DNA ends, and the environment of histone tails. Therefore, the damaged DNA structure can influence both the amount and the length of PAR synthesized by PARP2.
Mutations in the N-terminal peptide (Ser-Thr to Ala-Gly substitution) of the coat protein (CP) of potato virus X (PVX-ST) render its genomic RNA translationally competent, unlike in the wild-type PVX virions. Consequently, RNA within the PVX-ST virions can be translated without additional triggers (such as phosphorylation or interaction with the triple gene block 1 protein), unlike the encapsidated RNA of the wild-type virus. Comprehensive structural analysis using molecular dynamics (MD), small-angle X-ray scattering (SAXS), and tritium planigraphy revealed differences in the virion organization. The mutations were shown to increase hydrophobicity and induce partial folding of the N-terminal peptides. This triggers structural rearrangement in the PVX-ST virion: packing density of the coat proteins within the helical capsid is altered. This conclusion is supported by the SAXS data, increased accessibility for tritium labeling of the key CP domains (including the RNA-binding region), and reduced stability against the action of the sodium dodecyl sulfate detergent. The obtained results provide explanation for the mechanism by which the encapsidated RNA of the PVX-ST mutant becomes accessible to ribosomes. This mechanism is associated with structural rearrangement of the N-terminal coat protein peptide and change in the packing density of the helical capsid.
This article reviews biosynthesis of the valuable fat-soluble compounds with antioxidant activity, in particular vitamin E isomers and carotenoids, in yeast cells. Main genetic engineering approaches to increase microbial production of these substances are described. The main innovative strategies for subcellular separation of synthesis, storage, and recovery of lipophilic compounds are discussed, and examples of cell morphology engineering importance of are shown.
Patients with coronary and cerebral atherosclerosis are characterized by increased levels of total serum calcium, ionized calcium, and phosphate, against a background of reduced levels of total serum protein and albumin. Here we aimed to develop a rapid diagnostic assay for mineral homeostasis disorders, based on assessing capacity of the acidic plasma proteins to bind excess calcium and phosphate ions. Plasma from bony fish, amphibians, reptiles, birds, mice, and patients with myocardial infarction was incubated with excess concentrations of calcium and phosphate at 37°C for varying time periods. The following assay readouts were defined: (i) plasma optical density after supersaturation with calcium and phosphate ions, reflecting excessive formation of calciprotein particles (CPPs); and (ii) CPP concentration in plasma. CPPs were formed in all vertebrates. The most pronounced plasma calcification propensity was observed in the human and mouse plasma, suggesting an evolutionary significance of CPP formation as a mechanism for clearance of excess circulating calcium and phosphate ions in mammals. Among the 11 protocols of supersaturation with calcium and phosphate ions, stable increase in plasma optical density at 620 nm wavelength (normalized OD620, a measure of plasma calcification propensity) was achieved by adding solutions of CaCl2 (+2 mmol/L, +50 µL), Na2HPO4·12H2O (+2 mmol/L, +50 µL), and NaCl (+15.4 mmol/L, +20 µL) to plasma (80 µL). Increase in the normalized OD620 was consistently detected within 10 min from the reaction onset during incubation in a microplate shaker (37°C), with mild-to-moderate variability across the parallel or sequential measurements and between the different operators. These results support relevance of validating the developed diagnostic assay for assessing mineral homeostasis disorders in the expanded cohorts of patients with myocardial infarction and ischemic stroke.
Pyridoxal-5'-phosphate (PLP)-dependent D-amino acid transaminases (DATAs) catalyze stereoselective transfer of an amino group from a D-amino acid to an α-keto acid to form new D-amino acid and α-keto acid. These enzymes are found in bacteria and plants; they are responsible for the synthesis of D-amino acids and are incorporated into the nitrogen cycle. In general, the mechanism of D-transamination is similar to the known mechanism of transamination for aspartate aminotransferase: D-transamination reaction consists of two half-reactions with intermediate transfer of the amino group to the cofactor and formation of its reduced form, pyridoxamine-5'-phosphate. DATAs are characterized by broad substrate specificity and an open active site, which, however, does not affect their high stereoselectivity: no side L-products is detected in the DATA-catalyzed D-transamination. As in other PLP-dependent fold type IV transaminases, the functional unit of DATAs is a dimer. The active site is formed by amino acid residues of both subunits and binding of α-carboxylate group is crucial for proper substrate coordination. DATAs with promiscuous activity towards substrates without an α-carboxylate group, primary (R)-amines, have also been discovered and characterized. The promiscuous activity is achieved through the mobility of certain residues in the active site of DATAs. High stereoselectivity and stability of DATAs make then promising candidates for multienzyme cascade processes as biocatalysts of the (R)-stereoselective amination stage. Open configuration of active site makes binding and conversion of bulk non-natural substrates possible. The review describes in detail properties, structure, and relationships of DATAs from two currently known groups differing in organization of their active sites. The prospects for biotechnological applications of DATAs are discussed as well.
Macrophages are a heterogeneous cell population whose functional diversity is formed during their maturation and depends on factors of the microenvironment after their migration into the bloodstream or tissues. One such factor is the pro-inflammatory protein cyclophilin A (CypA, 18 kDa). Using a model of early human monocytic THP-1 cells, it was shown that recombinant human CypA (rhCypA) exerts a differentiating effect on these cells, inducing their maturation, adhesion, and spreading. Under the effect of rhCypA, the THP-1 cells developed an actin cytoskeleton characteristic of motile cells with numerous pseudopodia and podosomes, which ensure tight adhesion of the cells to the substrate and determine their migratory capabilities. Combination of low concentrations of rhCypA and other activators (phorbol myristate acetate) showed an additive effect and ensured effective monocyte differentiation. It was shown that rhCypA, along with other pro-inflammatory factors (IFNγ, TNFα), promotes cell fusion and induces formation of multinucleated macrophages, which are formed during osteoclast maturation under normal conditions as well as during granuloma formation in chronic inflammation (tuberculosis, Crohn's disease). Multinucleated giant cells have significantly higher functional activity (phagocytosis, bactericidal, and pro-inflammatory activity) compared to the mononuclear forms. The study showed that rhCypA enhances expression of the CD147 molecule, an integral functional regulator of CD29 and CD98 molecules involved in the processes of cell adhesion and fusion. Elevated doses of CypA cause deterioration in macrophages, inducing their apoptosis, which may play a role in regulation of the immune response. The findings of this study determined the mechanisms by which secreted CypA mediates monocyte differentiation and maturation, as well as it showed functional role of macrophages in the development of the immune response, which could facilitate further development of therapeutic approaches for the treatment of infectious, autoimmune, and other diseases.
Systemic blockade of proinflammatory cytokines such as IL-1, TNF, and IL-6 using therapeutic antibodies has proven effective in treating a wide range of autoimmune and other chronic inflammatory diseases. However, such blockade also suppresses non-redundant protective and homeostatic functions of cytokines, leading to a number of undesirable side effects. In this study, a novel bispecific mini-antibody featuring modules targeting human TNF and CD14 demonstrated efficacy in controlling TNF secretion from human peripheral blood monocytes. Administration of this antibody protected humanized TNF mice from lethal hepatotoxicity induced by a combination of LPS and D-galactosamine.
Filamin C is an adapter protein involved in the regulation of cytoskeleton; it interacts with more than 90 protein partners, including small heat shock proteins (sHsps). However, the details of filamin C interaction with sHsps remain poorly characterized. Here, we used immunochemistry methods, size-exclusion chromatography, native gel electrophoresis, and chemical crosslinking to investigate the interactions of a long C-terminal fragment of filamin C containing immunoglobulin (Ig)-like domains 19-24 (FLNC19-24) with sHsps. Out of five analyzed sHsps (HspB1, phosphorylation-mimicking 3D mutant of HspB1, HspB5, HspB6, HspB7, and HspB8), only HspB7 formed complexes with FLNC19-24. Taking into account that HspB7 interacted with the isolated Ig-like domain 24 and filamin fragments containing Ig-like domains 22-24 and 19-24, we concluded that HspB7 is a bona fide partner of filamin C. Selective binding of the α-crystallin domain of HspB7 with the Ig-like domain 24 induced dissociation of filamin dimers, which might promote filamin C translocation in the cell and facilitate the repairs of damaged contractile apparatus.
In recent years, reconstructive surgery strategies have been supplemented with innovative approaches aimed at developing tissue-engineered structures using autologous tissues and biodegradable scaffolds and matrices, the purpose of which is to reduce the risk of postoperative complications, on the one hand, while ensuring accelerated restoration of the structure and functions of organs, on the other hand. The review systematizes modern scientific trends in the field of tissue engineering of the urethra, focused on creation of biocompatible and functionally active tissue-engineered structures using achievements of cellular technologies and materials science. Particular attention is paid to describing the mechanisms and ways of forming a complete and functional three-dimensional structure of the urethra, including the use of organoids. It is expected that this strategy will contribute to the development of personalized therapeutic approaches and improved clinical outcomes in the patients with urological diseases.
Sphingosine-1-phosphate (S1P) is one of the most extensively studied bioactive signaling molecules of sphingolipid metabolism, which plays a pivotal role in regulating numerous processes in the central nervous system and immune system. Acting as an extracellular ligand for five subtypes of G-protein-coupled receptors (S1PR1-S1PR5) as well as an intracellular metabolic mediator, S1P controls lymphocyte migration, blood-brain barrier permeability, survival and differentiation of oligodendrocytes, reactivity of astrocytes and microglia, and balance between inflammation, neurodegeneration, and neuroprotection. In pathogenesis of the demyelinative diseases, particularly multiple sclerosis, disruption of the "sphingolipid rheostat" is observed - a shift toward predominance of pro-apoptotic ceramides and relative decrease in the S1P levels, which promotes prevalence of the neuroinflammatory and neurodegenerative processes over remyelination. This review summarizes current data on the structure, metabolism, and intra- and extracellular signaling pathways of S1P, its dual role under physiological conditions and in multiple sclerosis, and analyzes approaches to pharmacological modulation of S1P signaling pathways, highlighting the prospects of selective targeted therapy aimed at immunomodulation, neuroprotection, and stimulation of remyelination.
Ecological adaptations of a species can be shaped by its repertoire of gene variants. The black garden ant Lasius niger shows high level of CYP9E duplication. In contrast to its congener, the jet ant L. fuliginosus, it exhibits tolerance toward fungus-infected aphids. In these two species, we compared expression of a subset of CYP9E genes, potentially involved in mycotoxin metabolism. No significant differences in expression were found. Similarly to L. niger, the jet ant has six copies of these genes, grouping pairwise on the phylogenetic tree with their L. niger counterparts. Beyond the gene subset targeted in the expression study, we found multiple CYP9E genes in the genomes of L. niger, L. fuliginosus, and - fewer by a third - in the outgroup Formica rufa, suggesting CYP9E amplification as an ancestral trait of the genus Lasius or a more basal clade.
Periodic hypoxia is a condition characterized by alternating episodes of oxygen deprivation (hypoxia) and periods of normal or elevated oxygen levels (reoxygenation). Depending on severity and duration of exposure, periodic hypoxia can activate both protective and pathological mechanisms. The aim of this study was to evaluate dependence of the effects of acute and periodic hypoxia on sex and age of rats. Male and female Wistar rats aged 2 and 4 months were used. The animals were exposed to normobaric hypoxia (8% O2, 2 h) either once or daily for 5 consecutive days. Subsequently, changes in body weight, activity and content of antioxidant system enzymes in blood plasma, as well as expression levels of the HIF-1α, GPx4, BDNF, and caspase-3 genes in the frontal cortex, hippocampus, and striatum of the brain were assessed. It was shown that daily hypoxia exposure leads to the decrease in body weight in both male and female rats of both ages. In the males, age-dependent changes in activity of antioxidant enzymes and increased expression of hypoxia marker genes in the brain were observed after a single hypoxia exposure. After multiple exposures, the recorded parameters did not differ from the control values. In the females, exposure to hypoxia did not affect activity of antioxidant enzymes, and increase in the expression of the studied genes was observed only after five daily exposures to hypoxia. The experiments revealed significant differences in the response to acute and periodic hypoxia exposure between male and female rats. These data should be considered when developing experimental models of periodic hypoxia and studying the mechanisms of adaptive and pathological reactions of the body to repeated hypoxia/reoxygenation episodes.
Brain-derived neurotrophic factor (BDNF) is widely recognized as a critical molecule for the survival, growth, and maintenance of neurons in both the central and peripheral nervous systems, as well as for the development of cognitive abilities and emotions. However, recent studies have shown that, in addition to its role as a universal brain "fertilizer", BDNF acts as a metabotrophin linking neuronal signaling with systemic metabolism. BDNF serves as a key factor that integrates the body's response to stress, physical activity, and food intake with cellular mechanisms underlying neural plasticity and normal brain function. The review presents evidence supporting BDNF as a bidirectionally metabolic "bridge": body metabolism controls BDNF production in the brain, while brain BDNF regulates body metabolism. Disruption of this regulatory axis is associated with a broad range of neurological and somatic disorders, as well as their comorbidities. Cellular mechanisms associated with disruptions in BDNF functions are explored in detail through the example of alcohol dependence, a condition characterized by both impaired brain signaling and somatic pathologies accompanied by metabolic changes.
Presynaptic nerve terminals contain a large number of vesicles filled with neurotransmitters, whose release ensures signal transmission from the presynaptic neuron to the postsynaptic cell. Despite their morphological homogeneity, synaptic vesicles (SVs) are functionally heterogeneous and are organized into distinct groups (pools) that differ in their ability for exocytosis and mobilization, recycling kinetics, and protein composition. In addition to the classic pools - the readily releasable pool (RRP), recycling pool, and reserve pool - other populations have been identified, including spontaneously recycling vesicles, vesicles of resting pool and superpool. Vesicles from different pools engage in different modes of exocytosis and endocytosis, and the extent of interpool mixing varies depending on the synapse type and physiological or pathological conditions. Changes in the organization of SV pools underlie multiple forms of synaptic plasticity. Furthermore, SV cycling is a target of several pharmacological agents, and its disruption plays a significant role in the pathogenesis of neurodegenerative diseases. This article is a systematic review of SV pools, their organizational features in central and peripheral synapses, and implications of changes in the structure of SV pools in synaptic plasticity, action of drugs, and development of neurological disorders.