Pathological aggregation of α-synuclein is a key event in the development of synucleinopathies, such as Parkinson’s disease and Lewy body dementia. Currently, no effective disease-modifying therapy is available, necessitating the search for new therapeutic agents. One promising strategy involves the use of low-molecular-weight compounds capable of inhibiting the formation of toxic protein aggregates. This study evaluates the anti-aggregation properties of EC3222x, a conjugate of pharmacophoric fragments of amantadine and a fluorinated derivative of tetrahydro-γ-carboline. α-Synucleinopathy was modeled in the SH-SY5Y neuroblastoma cell line by transfection with a plasmid vector encoding the mutant human α-synuclein A53T protein. EC3222x at a concentration of 1 µM reduced the number of cells with α-synuclein A53T aggregates. Its efficacy was comparable to that of SynuClean-D and Buntanetap, known inhibitors of α-synuclein aggregation. Treatment with EC3222x reduced both the level of diffusely distributed intracellular α-synuclein and the formation of mature fibrillar aggregates and large aggresomes. Importantly, EC3222x did not affect the accumulation of another aggregation-prone protein, TDP-43, in a similar cellular model, indicating its specificity for α-synuclein. These findings suggest that EC3222x may represent a promising candidate for the development of therapeutic agents targeting synucleinopathies.
Epilepsy is a severe chronic condition that remains pharmacoresistant in approximately 30
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.
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
Parkinson’s disease is associated with amyloid aggregation of alpha-synuclein, which could be affected by the proteins of the SARS-CoV-2 coronavirus, possibly accelerating and provoking neurodegeneration. The purpose of this work was to compare the effects of the N-protein and the receptor binding domain (RBD) of the S protein on fibrillization of the alpha-synuclein preparation produced using an original technique that excludes presence of non-native forms of alpha-synuclein that alter kinetics of the process. Presence of an elongated form of alpha-synuclein in the previously studied protein preparations is associated with the erroneous reading of the rare for E. coli TGA stop codon in the pET33b(+) expression plasmid as tryptophan, which led to the continued translation to the next stop codon. To prevent this effect, a new plasmid design was suggested with replacement of the original stop codon with a double stop codon TAA, which made it possible to obtain a homogeneous protein preparation without the admixture of alpha-synuclein with increased molecular weight. It has been shown that the N-protein is able to accelerate alpha-synuclein fibrillization, while the RBD of the S protein inhibits aggregation. According to the electron microscopy data, structure of the fibrils formed in the presence of viral proteins is also different. The obtained data are important for understanding the mechanisms of development of post-covid synucleinopathies, as well as consequences of vaccination with the viral proteins.
Dynamics of glial activity changes in the subacute and chronic stages of ischemic stroke after small focal injuries remains poorly understood due to complexity of the long-term animal monitoring and data interpretation. The aim of this study was to assess relationship between the delayed morphological changes in nervous tissue after experimental stroke and lesion parameters determined in vivo at various time points. For this purpose, photothrombotic ischemia of the cerebral cortex was induced in the C57BL/6J-Tg(Thy1-GCaMP6f)GP5.17Dkim/J mice, which express fluorescent calcium sensor protein GCaMP6f in cortical neurons. Lesion (ischemic core) size was determined using wide-field optical imaging (WFOI) through a cranial window via the GCaMP6f fluorescence at 3 min, 1 day, and 7 days post-photothrombosis. On day 19, brain sections were analyzed using Nissl staining and immunohistochemistry for microglial (Iba1) and astrocytic (GFAP) markers. It was found that the signs of neuroinflammation – changes in glial cell morphology and quantity – persist in the perifocal region even 19 days after ischemia induction, despite the small lesion volume. A significant linear relationship between microglial nuclear area and lesion size on day 7 was identified. Conversely, no significant correlation was found between the lesion sizes determined in the hyperacute phase (3 min) and acute phase (1 day) and cellular parameters (cell count, morphometric parameters). This indicates that the lesion formation in the acute phase is dynamic, and only the lesion size after its stabilization influences long-term stroke outcomes. Absence of a correlation between the delayed glial changes and ischemic core size during the hyperacute and acute phases suggests that therapeutic window for interventions modulating glial activity may extend to the later period after stroke, even with small lesion size. The results also allow us to conclude that it is not necessary to make an amendment for the initial lesion size in the studies of delayed neuroglial processes in preclinical models. In turn, the correlation between the lesion size on day 7 and microglial cell nucleus area on day 19 demonstrates that the lesion size at the end of the acute phase may be one of the prognostic factors for effectiveness of the post-stroke therapy.
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.
Allergic contact dermatitis (ACD) is a chronic inflammatory skin disorder the development of which is driven by allergen sensitization in peripheral lymphoid organs and local cutaneous inflammation. Lymphotoxin (LT) and its receptor LTβR are critical for lymphoid organogenesis and immune regulation in barrier tissues, but their role in ACD pathogenesis remains incompletely defined. This study aimed to delineate differential contribution of the LTβR-dependent signaling in oxazolone-induced dermatitis. We examined Lta knockout (Lta KO) mice, which lack both soluble LTα3 and membrane-bound isoforms LTα1β2/LTα2β1, and the Ltbr knockout (Ltbr KO) mice, both of which lack lymph nodes. ACD was induced by repeated oxazolone application to ear skin, with assessment of clinical severity, inflammation-associated gene expression, serum IgE levels, and immune cell composition in blood and spleen. Contrary to previous reports, the Lta KO mice developed dermatitis comparable to the wild-type (WT) mice, with elevated IgE production. In contrast, the Ltbr KO mice were substantially protected from the disease, exhibiting attenuated clinical inflammation, reduced ear swelling, and decreased Tslp expression in the lesional skin at the background of a lower proportion of circulating CD4+ T cells. These findings indicate that LTβR-dependent signaling is pathogenic in allergic skin inflammation, while LTα-mediated pathways are dispensable, suggesting a potential role for the other LTβR ligand, LIGHT, in ACD pathogenesis. Notably, ACD developed even in the absence of lymph nodes, highlighting the importance of local, skin-resident LTβR-dependent mechanisms in the disease development.
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.
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.
Major histocompatibility complex class I (MHC I) plays a crucial role in immune functions. This complex typically binds short fragments of protein chains, 8-9 amino acid residues in length, referred to as epitopes. In this study, we investigated differences between the peptides that bind to this complex (dataset N1) and those that do not (dataset N0). To compare the datasets N1 and N0, Z-score analysis using the Z-score function was applied to identify statistically significant differences in physicochemical properties under study: aliphatic index (αi), charge (Zi), hydrophobicity (Hi), isoelectric point (pIi), molecular weight (Mi), and instability index (IIi). All properties except for the instability index depend solely on amino acid composition of the peptides and not on the sequence-specific features. For the evaluated physicochemical properties, the Z-score values indicated no significant differences between the datasets N1 and N0. Maximum Z-score values were 0.30 for the aliphatic index and 0.29 for hydrophobicity. The most robust and reliable separation between the datasets N1 and N0 was achieved using the r-value method, yielding classification accuracy of approximately 70
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.
The primary role of sterols in the cell is to support plasma membrane function, and for this reason their concentration in this compartment is the highest among all cell membranes. In the yeast Saccharomyces cerevisiae, sterol transport between membranes is mediated by proteins of Osh and Lam families. The Lam1-Lam4 proteins are reported to transport sterols passively from plasmalemma to endoplasmic reticulum. The Lam5-Lam6 proteins transport sterols at the ER interface with vacuoles and mitochondria. Deletion of the LAM family genes does not impair cell growth under standard conditions, which makes their biological role unclear. We hypothesized that the Lam family proteins may play a role in yeast sporulation, as the spore plasma membrane is formed de novo from the ER-derived vesicles, which contain less sterol than the plasma membrane, necessitating sterol transport into the newly forming spore plasma membrane. To test this hypothesis, we generated diploid strains with the LAM1-LAM4 and LAM5-LAM6 deletions. We demonstrated that double deletion of the LAM5-LAM6 genes reduced both percentage of the sporulating cells and number of the spores per ascus. Conversely, deletion of the LAM1-LAM4 genes reduced proportion of the full asci but did not inhibit sporulation initiation. We demonstrated that deletion of the LAM1-LAM4 genes induces cell wall thickening and structural defects. Clusters of osmiophilic granules were detected at the cell wall surface of these spores. Spores with the deletions of the LAM family genes demonstrated reduced resistance to heat shock and alkali. Taken together, our data indirectly support our hypothesis and point out that sterol transport by the LAM family proteins is necessary for the sterol redistribution during sporulation.
Ultracentrifugation (UC) has long been considered the “gold standard” for extracellular vesicle (EV) isolation. However, due to its drawbacks such as high cost of an ultracentrifuge and rotors, time-consuming and labor-intensive protocol, low yield considering initial biofluid volume and low throughput, development of new EV isolation approaches is still ongoing. Here we compare three methods for isolating the most studied EV subtype, small extracellular vesicles (sEVs), from human plasma: ultracentrifugation (UC), express asymmetric depth filtration (ExADFi), and anti-CD9 immunoaffinity capture (AS-CD9) with focus on their Raman and proteomic profiles. For all three methods, purity and quality of the sEV isolation were assessed based on the level of contamination of the sEV fraction with major plasma proteins such as albumin and apolipoproteins (APOA1, APOH, APOA4, APOC2, APOC1, and APOC4). UC showed the highest ratio of protein to nanoparticle concentration. AS-CD9 and ExADFi provided comparable to UC purity and levels of non-vesicular contaminants with AS-CD9 requiring minimal time and labor. ExADFi showed characteristics including purity of the sEV samples, yield, and isolation time that is between the UC and AS-CD9 methods. Raman spectroscopy provided more details about characteristics of the isolated sEVs and confirmed differences observed in the proteomic profiles. The findings demonstrate that the AS-CD9 and ExADFi methods could be appropriate substitutes of the classical UC-based isolation method and be chosen depending on the final requirements and use of the purified sEVs such as further functional and biomarker studies.
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.
Brain aging is a physiological process characterized by various neurodegenerative manifestations, largely driven by mitochondrial dysfunctions, including changes in mitochondrial metabolism and dynamics. Conflicting reports in the literature regarding mitochondrial fusion and fission in the human cerebral cortex during aging underscore the need to elucidate the mechanisms of this dysfunction. The aim of this study was to assess features of mitochondrial dynamics in the large pyramidal neurons of the human motor cortex during aging. The study was conducted on autopsy material from the motor cortex of individuals aged 75 years and older. The control group consisted of similar material from individuals aged 35-44 years who died from sudden cardiac death. Intensity of immunohistochemical staining for TOMM20, Drp1, Mfn1, Mfn2, and Opa1 proteins in the large pyramidal neurons of the human motor cortex was evaluated. Decrease in the staining intensity of TOMM20 and Opa1 markers and increase in the staining intensity of the Drp1 marker were observed, indicating enhanced mitochondrial fragmentation in the pyramidal neurons of layer V of the motor cortex, possibly associated with reduction in the mitochondrial pool volume due to dysfunction in the mitochondrial fusion process, which impedes organelle growth.
Hepatitis B virus (HBV) infects human hepatocytes, causing acute or chronic liver infection. Chronic HBV infection leads to progressive liver damage, potentially resulting in cirrhosis or hepatocellular carcinoma. One promising antiviral strategy involves activating cytidine deaminases of the APOBEC/AID family, which could induce mutational degradation of HBV. Using a CRISPRa-based transcriptional activation system with modified sgRNAs, we investigated antiviral and oncogenic effects of the activating genes encoding APOBEC3C, APOBEC3D, and APOBEC3H.
Air pollution remains a major environmental challenge, largely driven by urban dust composed of suspended solid particles of diverse origins and chemical compositions. Particulate matter (PM2.5) and ultrafine urban dust nanoparticles (NPs), with diameters smaller than 2.5 μm and 100 nm, respectively, pose a particular threat to human health. In this study, we present the first evidence that NPs induce pro-inflammatory activation of human bronchial epithelial cells. Exposure to non-cytotoxic concentrations of NPs led to a significant increase in the mRNA levels of pro-inflammatory markers IL-8, IL-1β, IL-6, and ICAM-1, accompanied by increased secretion of the cytokines IL-8 and IL-6. Heat treatment of NPs, which removed their organic components, completely abolished their ability to stimulate cytokine secretion. NP-induced upregulation of pro-inflammatory gene expression depended on both surface-adsorbed organic compounds and inorganic particle constituents.
