Electrochemical Nanopipette Sensor for In Vitro/In Vivo Detection of Cu2+ Ions.

In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.

Scanning Ion-Conductance Microscopy for Studying ß-Amyloid Aggregate Formation on Living Cell Surfaces.

ß-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of ß-amyloid (Aß) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss, etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules and consequently alterations of cell mechanical properties. The effect of amyloid oligomers on cells' Young's modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on the local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young's modulus mapping, and confocal imaging of Aß aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aß leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aß aggregates on living cell surfaces.

Electrochemical Analysis in Studying ß-Amyloid Aggregation.

ß-amyloid (Aß) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aß aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aß aggregation have intensively being investigated using synthetic Aß peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aß aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aß (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aß oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aß, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aß are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aß aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.

Switching On/Off Amyloid Plaque Formation in Transgenic Animal Models of Alzheimer's Disease.

A hallmark of Alzheimer's disease (AD) are the proteinaceous aggregates formed by the amyloid-beta peptide (Aß) that is deposited inside the brain as amyloid plaques. The accumulation of aggregated Aß may initiate or enhance pathologic processes in AD. According to the amyloid hypothesis, any agent that has the capability to inhibit Aß aggregation and/or destroy amyloid plaques represents a potential disease-modifying drug. In 2023, a humanized IgG1 monoclonal antibody (lecanemab) against the Aß-soluble protofibrils was approved by the US FDA for AD therapy, thus providing compelling support to the amyloid hypothesis. To acquire a deeper insight on the in vivo Aß aggregation, various animal models, including aged herbivores and carnivores, non-human primates, transgenic rodents, fish and worms were widely exploited. This review is based on the recent data obtained using transgenic animal AD models and presents experimental verification of the critical role in Aß aggregation seeding of the interactions between zinc ions, Aß with the isomerized Asp7 (isoD7-Aß) and the α4ß2 nicotinic acetylcholine receptor.

Natural Isoforms of Listeria monocytogenes Virulence Factor Inlb Differ in c-Met Binding Efficiency and Differently Affect Uptake and Survival Listeria in Macrophage.

Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms on L. monocytogenes uptake and intracellular proliferation in human macrophages. Three isoforms of the receptor binding domain (idInlB) were derived from phylogenetically distinct L. monocytogenes strains belonging to the highly virulent CC1 (idInlBCC1), medium-virulence CC7 (idInlBCC7), and low-virulence CC9 (idInlBCC9) clonal complexes. The constant dissociation increased in the order idInlBCC1 << idInlBCC7 < idInlBCC9 for interactions with c-Met, and idInlBCC1 ≈ idInlBCC7 < idInlBCC9 for interactions with gC1q-R. The comparison of uptake and intracellular proliferation of isogenic recombinant strains which expressed full-length InlBs revealed that the strain expressing idInlBCC1 proliferated in macrophages twice as efficiently as other strains. Macrophage pretreatment with idInlBCC1 followed by recombinant L. monocytogenes infection disturbed macrophage functions decreasing pathogen uptake and improving its intracellular multiplication. Similar pretreatment with idInlBCC7 decreased bacterial uptake but also impaired intracellular multiplication. The obtained results demonstrated that InlB impaired macrophage functions in an idInlB isoform-dependent manner. These data suggest a novel InlB function in L. monocytogenes virulence.

Activation of Purine Biosynthesis Suppresses the Sensitivity of E. coli gmhA Mutant to Antibiotics.

Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis (ΔgmhA) prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress. We found that the derepression of purine biosynthesis (∆purR) normalizes the metabolic equilibrium in PPP in ΔgmhA mutants, suppressing the negative effects of gmhA mutation likely via the over-expression of the glycine-serine pathway that is under the negative control of PurR and might be responsible for the enhanced synthesis of NADPH and glutathione. Consistently, the activity of the soxRS system, as well as the level of glutathionylation and oxidation of proteins, indicative of oxidative stress, were reduced in the double ΔgmhAΔpurR mutant compared to the ΔgmhA mutant.

Molecular Mechanism of Zinc-Dependent Oligomerization of Alzheimer's Amyloid-ß with Taiwan (D7H) Mutation.

Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

Changes in Hemoglobin Properties in Complex with Glutathione and after Glutathionylation.

Hemoglobin is the main protein of red blood cells that provides oxygen transport to all cells of the human body. The ability of hemoglobin to bind the main low-molecular-weight thiol of the cell glutathione, both covalently and noncovalently, is not only an important part of the antioxidant protection of red blood cells, but also affects its affinity for oxygen in both cases. In this study, the properties of oxyhemoglobin in complex with reduced glutathione (GSH) and properties of glutathionylated hemoglobin bound to glutathione via an SS bond were characterized. For this purpose, the methods of circular dichroism, Raman spectroscopy, infrared spectroscopy, tryptophan fluorescence, differential scanning fluorimetry, and molecular modeling were used. It was found that the glutathionylation of oxyhemoglobin caused changes in the secondary structure of the protein, reducing the alpha helicity, but did not affect the heme environment, tryptophan fluorescence, and the thermostability of the protein. In the noncovalent complex of oxyhemoglobin with reduced glutathione, the secondary structure of hemoglobin remained almost unchanged; however, changes in the heme environment and the microenvironment of tryptophans, as well as a decrease in the protein's thermal stability, were observed. Thus, the formation of a noncovalent complex of hemoglobin with glutathione makes a more significant effect on the tertiary and quaternary structure of hemoglobin than glutathionylation, which mainly affects the secondary structure of the protein. The obtained data are important for understanding the functioning of glutathionylated hemoglobin, which is a marker of oxidative stress, and hemoglobin in complex with GSH, which appears to deposit GSH and release it during deoxygenation to increase the antioxidant protection of cells.

Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Novel Hydroxamic Acids Containing Aryl-Substituted 1,2,4- or 1,3,4-Oxadiazole Backbones and an Investigation of Their Antibiotic Potentiation Activity.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc amidase that catalyzes the second step of the biosynthesis of lipid A, which is an outer membrane essential structural component of Gram-negative bacteria. Inhibitors of this enzyme can be attributed to two main categories, non-hydroxamate and hydroxamate inhibitors, with the latter being the most effective given the chelation of Zn2+ in the active site. Compounds containing diacetylene or acetylene tails and the sulfonic head, as well as oxazoline derivatives of hydroxamic acids, are among the LpxC inhibitors with the most profound antibacterial activity. The present article describes the synthesis of novel functional derivatives of hydroxamic acids-bioisosteric to oxazoline inhibitors-containing 1,2,4- and 1,3,4-oxadiazole cores and studies of their cytotoxicity, antibacterial activity, and antibiotic potentiation. Some of the hydroxamic acids we obtained (9c, 9d, 23a, 23c, 30b, 36) showed significant potentiation in nalidixic acid, rifampicin, and kanamycin against the growth of laboratory-strain Escherichia coli MG1655. Two lead compounds (9c, 9d) significantly reduced Pseudomonas aeruginosa ATCC 27853 growth in the presence of nalidixic acid and rifampicin.

Synthesis of the Indole-Based Inhibitors of Bacterial Cystathionine γ-Lyase NL1-NL3.

Bacterial cystathionine γ-lyase (bCSE) is the main producer of H2S in pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, etc. The suppression of bCSE activity considerably enhances the sensitivity of bacteria to antibiotics. Convenient methods for the efficient synthesis of gram quantities of two selective indole-based bCSE inhibitors, namely (2-(6-bromo-1H-indol-1-yl)acetyl)glycine (NL1), 5-((6-bromo-1H-indol-1-yl)methyl)- 2-methylfuran-3-carboxylic acid (NL2), as well as a synthetic method for preparation 3-((6-(7-chlorobenzo[b]thiophen-2-yl)-1H-indol-1-yl)methyl)- 1H-pyrazole-5-carboxylic acid (NL3), have been developed. The syntheses are based on the use of 6-bromoindole as the main building block for all three inhibitors (NL1, NL2, and NL3), and the designed residues are assembled at the nitrogen atom of the 6-bromoindole core or by the substitution of the bromine atom in the case of NL3 using Pd-catalyzed cross-coupling. The developed and refined synthetic methods would be significant for the further biological screening of NL-series bCSE inhibitors and their derivatives.

Synthetic, Cell-Derived, Brain-Derived, and Recombinant ß-Amyloid: Modelling Alzheimer's Disease for Research and Drug Development.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, characterised by the accumulation of senile plaques and tau tangles, neurodegeneration, and neuroinflammation in the brain. The development of AD is a pathological cascade starting according to the amyloid hypothesis with the accumulation and aggregation of the ß-amyloid peptide (Aß), which induces hyperphosphorylation of tau and promotes the pro-inflammatory activation of microglia leading to synaptic loss and, ultimately, neuronal death. Modelling AD-related processes is important for both studying the molecular basis of the disease and the development of novel therapeutics. The replication of these processes is often achieved with the use of a purified Aß peptide. However, Aß preparations obtained from different sources can have strikingly different properties. This review aims to compare the structure and biological effects of Aß oligomers and aggregates of a higher order: synthetic, recombinant, purified from cell culture, or extracted from brain tissue. The authors summarise the applicability of Aß preparations for modelling Aß aggregation, neurotoxicity, cytoskeleton damage, receptor toxicity in vitro and cerebral amyloidosis, synaptic plasticity disruption, and cognitive impairment in vivo and ex vivo. Further, the paper discusses the causes of the reported differences in the effect of Aß obtained from the sources mentioned above. This review points to the importance of the source of Aß for AD modelling and could help researchers to choose the optimal way to model the Aß-induced abnormalities.

Docking and Molecular Dynamics-Based Identification of Interaction between Various Beta-Amyloid Isoforms and RAGE Receptor.

Beta-amyloid peptide (Aß) is a ligand associated with RAGE (Advanced glycosylation end product-specific receptor). Aß is translocated in complexes with RAGE from the blood to brain across the blood-brain barrier (BBB) by transcytosis. Aß and its isoforms are important factors in the Alzheimer's disease (AD) pathogenesis. However, interaction with RAGE was previously studied for Aß but not for its isoforms. The present study has been directed at identifying the key interaction interfaces between RAGE and Aß isoforms (Aß40, Aß42, phosphorylated and isomerized isoforms pS8-Aß42, isoD7-Aß42). Two interfaces have been identified by docking: they are represented by an extended area at the junction of RAGE domains V and C1 and a smaller area linking C1 and C2 domains. Molecular dynamics (MD) simulations have shown that all Aß isoforms form stable and tightly bound complexes. This indicates that all Aß isoforms potentially can be transported through the cell as part of a complex with RAGE. Modeling of RAGE interaction interfaces with Aß indicates which chemical compounds can potentially be capable of blocking this interaction, and impair the associated pathogenic cascades. The ability of three RAGE inhibitors (RAP, FPS-ZM1 and RP-1) to disrupt the RAGE:Aß interaction has been probed by docking and subsequently the complexes' stability verified by MD. The RP-1 and Aß interaction areas coincide and therefore this inhibitor is very promising for the RAGE:Aß interaction inhibition.

Distinct Effects of Beta-Amyloid, Its Isomerized and Phosphorylated Forms on the Redox Status and Mitochondrial Functioning of the Blood-Brain Barrier Endothelium.

The Alzheimer's disease (AD)-associated breakdown of the blood-brain barrier (BBB) promotes the accumulation of beta-amyloid peptide (Aß) in the brain as the BBB cells provide Aß transport from the brain parenchyma to the blood, and vice versa. The breakdown of the BBB during AD may be caused by the emergence of blood-borne Aß pathogenic forms, such as structurally and chemically modified Aß species; their effect on the BBB cells has not yet been studied. Here, we report that the effects of Aß42, Aß42, containing isomerized Asp7 residue (iso-Aß42) or phosphorylated Ser8 residue (p-Aß42) on the mitochondrial potential and respiration are closely related to the redox status changes in the mouse brain endothelial cells bEnd.3. Aß42 and iso-Aß42 cause a significant increase in nitric oxide, reactive oxygen species, glutathione, cytosolic calcium and the mitochondrial potential after 4 h of incubation. P-Aß42 either does not affect or its effect develops after 24 h of incubation. Aß42 and iso-Aß42 activate mitochondrial respiration compared to p-Aß42. The isomerized form promotes a greater cytotoxicity and mitochondrial dysfunction, causing maximum oxidative stress. Thus, Aß42, p-Aß42 and iso-Aß42 isoforms differently affect the BBBs' cell redox parameters, significantly modulating the functioning of the mitochondria. The changes in the level of modified Aß forms can contribute to the BBBs' breakdown during AD.

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone-2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2-Polyamine complex formation. Thus, the (OAZ1)2-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds-2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin.

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin-myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.

Synthetic Optimizations for Gram-Scale Preparation of 1-O-Methyl d-Glycero-α-d-gluco-heptoside 7-Phosphate from d-Glucose.

Heptose phosphates-unique linkers between endotoxic lipid A and O-antigen in the bacterial membrane-are pathogen-associated molecular patterns recognized by the receptors of the innate immune system. Understanding the mechanisms of immune system activation is important for the development of therapeutic agents to combat infectious diseases and overcome antibiotic resistance. However, in practice, it is difficult to obtain a substantial amount of heptose phosphates for biological studies due to the narrow scope of the reported synthetic procedures. We have optimized and developed an inexpensive and convenient synthesis for the first performed gram-scale production of 1-O-methyl d-glycero-α-d-gluco-heptoside 7-phosphate from readily available d-glucose. Scaling up to such amounts of the product, we have increased the efficiency of the synthesis and reduced the number of steps of the classical route through the direct phosphorylation of the O6,O7-unprotected heptose. The refined method could be of practical value for further biological screening of heptose phosphate derivatives.

Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model.

Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids.

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

The Dynamics of ß-Amyloid Proteoforms Accumulation in the Brain of a 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia among the elderly. Neuropathologically, AD is characterized by the deposition of a 39- to 42-amino acid long ß-amyloid (Aß) peptide in the form of senile plaques. Several post-translational modifications (PTMs) in the N-terminal domain have been shown to increase the aggregation and cytotoxicity of Aß, and specific Aß proteoforms (e.g., Aß with isomerized D7 (isoD7-Aß)) are abundant in the senile plaques of AD patients. Animal models are indispensable tools for the study of disease pathogenesis, as well as preclinical testing. In the presented work, the accumulation dynamics of Aß proteoforms in the brain of one of the most widely used amyloid-based mouse models (the 5xFAD line) was monitored. Mass spectrometry (MS) approaches, based on ion mobility separation and the characteristic fragment ion formation, were applied. The results indicated a gradual increase in the Aß fraction of isoD7-Aß, starting from approximately 8% at 7 months to approximately 30% by 23 months of age. Other specific PTMs, in particular, pyroglutamylation, deamidation, and oxidation, as well as phosphorylation, were also monitored. The results for mice of different ages demonstrated that the accumulation of Aß proteoforms correlate with the formation of Aß deposits. Although the mouse model cannot be a complete analogue of the processes occurring in the human brain in AD, and several of the observed parameters differ significantly from human values supposedly due to the limited lifespan of the model animals, this dynamic study provides evidence on at least one of the possible mechanisms that can trigger amyloidosis in AD, i.e., the hypothesis on the relationship between the accumulation of isoD7-Aß and the progression of AD-like pathology.