Molecular Basis of Complement C1q Collagen-Like Region Interaction with the Immunoglobulin-Like Receptor LAIR-1

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q’s CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.     

A comparative study of the thermal stability and reactions of CH2, CH3 and C2H5 species on the Pd(100) surface

Adsorbed CH₂, CH₂ and C₂H₅ moieties were produced on Pd(100) at 90 K by photoinduced dissociation of the corresponding iodo compounds, and their thermal reactions were established.This laboratory is a part of the Center of Catalysis, Surface and Material Science at the University of Szeged.     

The effects of a potassium overlayer on the reaction pathway of CH2 and C2H5 on Rh(111)

The effect of potassium on the reaction pathways of adsorbed CH₂ and C₂H₅ species on Rh(111) was investigated by means of reflection absorption infrared spectroscopy (RAIRS) and temperature programmed desorption (TDS). Hydrocarbon fragments were produced by thermal and photo-induced dissociation of the corresponding iodo compounds. Potassium adatoms markedly stabilized the adsorbed CH₂ and converted it into C₂H₄, the formation of which was not observed for K-free Rh(111). New routes of the surface reactions of C₂H₅ have been also opened in the presence of potassium, namely its transformation into butane and butene.     

The Interaction between Micrometer-size Particles and Flat Substrates: A Quantitative Study of Jump-to-Contact

The interaction force acting on an individual micrometer-size polystyrene particle near a flat, electrically conducting substrate has been measured by attaching the particle to an atomic force microscope cantilever. From the spatial dependence of the interaction force, the equations of motion governing a particle near the substrate can be determined. These considerations allow a prediction of the jump-to-contact distance of the particle as it approaches the substrate. This distance is measured as a function of particle radius and compared with predictions based on the relevant interaction force models.     

The Interaction between Micrometer-size Particles and Flat Substrates: A Quantitative Study of Jump-to-Contact

The interaction force acting on an individual micrometer-size polystyrene particle near a flat, electrically conducting substrate has been measured by attaching the particle to an atomic force microscope cantilever. From the spatial dependence of the interaction force, the equations of motion governing a particle near the substrate can be determined. These considerations allow a prediction of the jump-to-contact distance of the particle as it approaches the substrate. This distance is measured as a function of particle radius and compared with predictions based on the relevant interaction force models.     

2-(2-Hydroxy-5-nitrobenzylidene)-1,3-indanedione versus Fluorescein Isothiocyanate in Interaction with Anti-hFABP Immunoglobulin G1: Fluorescence Quenching,Secondary Structure Alteration and Binding Sites Localization

The first step in determining whether a fluorescent dye can be used for antibody labeling consists in collecting data on its physical interaction with the latter. In the present study, the interaction between the 2-(2-hydroxy-5-nitrobenzylidene)-1,3-indanedione (HNBID) dye and the IgG1 monoclonal mouse antibody anti-human heart fatty acid binding protein (anti-hFABP) has been investigated by fluorescence and circular dichroism spectroscopies and complementary structural results were obtained by molecular modeling. We have determined the parameters characterizing this interaction, namely the quenching and binding constants, classes of binding sites, and excited state lifetimes, and we have predicted the localization of HNBID within the Fc region of anti-hFABP. The key glycosidic and amino acid residues in anti-hFABP interacting with HNBID have also been identified. A similar systematic study was undertaken for the well-known fluorescein isothiocyanate fluorophore, for comparison purposes. Our results recommend HNBID as a valuable alternative to fluorescein isothiocyanate for use as a fluorescent probe for IgG1 antibodies.     

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.     

The skeletal isomerization of C4-C7 1-olefins over ferrierite and ZSM-5 zeolite catalysts

The skeletal isomerization of C₄-C₇ 1-olefins was studied on ferrierite (FER) and ZSM-5 (MFI) zeolites to elucidate the effect of the molecular distribution in zeolite pores on the selectivity foriso-olefin formation. Regardless of the difference in molecular length of 1-olefins, the FER zeolite showed high selectivity foriso-olefins, while the selectivity became slightly low at the skeletal isomerization of long olefin molecules. The drastic decrease in the selectivity of MFI zeolites by increasing the conversion is concurrently observed in the skeletal isomerization of C₄-C₇ 1-olefins. The high selectivity of FER zeolites is explained by their sparse distributions of olefin molecules in pores, which induces a high preference for monomolecular skeletal isomerization.     

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gα_s-coupled human adenosine receptors, A_2A (A_2AAR) and A_2B (A_2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A_2AAR and A_2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.     

Imaging the Detergency of Single Cotton Fibers with Confocal Microscopy: the Effect of Surfactants and Lipases

Detergency mechanisms of lipids from single cotton fibers were visualized by means of confocal laser scanning microscopy (CLSM). Fibers were soiled with different types of lipids: olive oil, lard and tri-C₁₀, and subsequently stained with the fluorescent probe Nile Red. A surfactant composition of 300 μM C₁₂E₆/LAS (1:2 mol%) was used to mimic the surfactants used in a common washing solution. It was evident from the captured image series that the different kinds of soiling were removed by different mechanisms by the surfactants, depending on the fluidity of the lipid. Roll-up was the main mechanism when removing olive oil, whereas emulsification (necking) and/or solubilization were observed in the removal of lard and tri-C₁₀. Only 20–25% of the olive oil remained after treatment with surfactants, which was much less compared to the solid fats where roughly 50% remained at end of treatment. The effect of adding lipases to the detergent formulation was clearly seen, both by an apparently higher rate of removal of olive oil but also using double injection when removing lard. A first injection of only surfactants removed a certain part of the lard as emulsion droplets stuck onto the fiber. A second injection of both lipases and surfactants was able to remove some of the preformed emulsion particles and reduce the overall remaining lard content on the cotton fiber.

Interaction between Melatonin and NO: Action Mechanisms,Main Targets,and Putative Roles of the Emerging Molecule NOmela

Melatonin (MEL), a ubiquitous indolamine molecule, has gained interest in the last few decades due to its regulatory role in plant metabolism. Likewise, nitric oxide (NO), a gasotransmitter, can also affect plant molecular pathways due to its function as a signaling molecule. Both MEL and NO can interact at multiple levels under abiotic stress, starting with their own biosynthetic pathways and inducing a particular signaling response in plants. Moreover, their interaction can result in the formation of NOmela, a very recently discovered nitrosated form of MEL with promising roles in plant physiology. This review summarizes the role of NO and MEL molecules during plant development and fruit ripening, as well as their interactions. Due to the impact of climate-change-related abiotic stresses on agriculture, this review also focuses on the role of these molecules in mediating abiotic stress tolerance and the main mechanisms by which they operate, from the upregulation of the entire antioxidant defense system to the post-translational modifications (PTMs) of important molecules. Their individual interaction and crosstalk with phytohormones and H₂S are also discussed. Finally, we introduce and summarize the little information available about NOmela, an emerging and still very unknown molecule, but that seems to have a stronger potential than MEL and NO separately in mediating plant stress response.     

The Role of hOGG1 C1245G Polymorphism in the Susceptibility to Lupus Nephritis and Modulation of the Plasma 8-OHdG in Patients with Systemic Lupus Erythematosus

We investigated whether the C1245G polymorphism of human 8-oxoguanine glycosylase 1 (hOGG1) gene confers the susceptibility to systemic lupus erythematosus (SLE) occurrence of lupus nephritis and affects the plasma level of 8-hydroxy-2''-deoxyguanosine (8-OHdG) in patients with SLE. A total of 45 healthy controls and 85 SLE patients were recruited. The C1245G polymorphism of the hOGG1 gene was determined by direct sequencing. The frequency of occurrence of the hOGG1 1245 GG genotype in SLE patients was 31.8% (27/85), which is lower than that of healthy controls of 53.3% (24/45). Thirty-three (33/85, 38.8%) SLE patients developed lupus nephritis. Significantly, SLE patients harboring the hOGG1 1245 GG genotype had a higher incidence to develop lupus nephritis than did those harboring the hOGG1 1245 CC or CG genotype (15/27, 55.6% vs.18/58, 31.0%, p = 0.031). Divided into subgroups, SLE patients harboring the hOGG1 1245 GG genotype had the highest plasma levels of 8-OHdG among patients with all genotypes, with regard to the coexistence of lupus nephritis (p = 0.020, ANOVA), including those with nephritis harboring the hOGG1 1245 CC or CG genotypes (p = 0.037), those without nephritis harboring the hOGG1 1245 GG genotype (p = 0.050), and those without nephritis harboring the hOGG1 1245 CC or CG genotype (p = 0.054). We conclude that the C1245G polymorphism of hOGG1 may be one of the factors that confer the susceptibility to lupus nephritis and modulate the plasma level of 8-OHdG in patients with SLE.     

Mutation in the CX3C Motif of G Protein Disrupts Its Interaction with Heparan Sulfate: A Calorimetric,Spectroscopic, and Molecular Docking Study

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention of attachment of virion to the host cell receptors. Collectively, this investigation suggests that mutation in the CX3C motif of G protein may likely improve the efficacy and safety of the RSV vaccine.     

The Effect of Ozone on Aflatoxin M1, Oxidative Stability,Carotenoid Content and the Microbial Count of Milk

In the current investigation, attempts were made to determine if ozone treatment can affect the aflatoxin M₁ (AFM₁) content of milk. Aflatoxin M₁ by a pre-determined concentration of 0.56 μg/Kg was added to milk. Milk samples, were then exposed to gaseous ozone (80 mg/min) in containers for durations of 0, 0.5, 1, 2, 5 and 10 min. The longer exposure time to ozone was more efficient in breaking down the AFM₁ in milk. Results indicated that AFM₁ was reduced by 50%, when milk was ozonated at for 5 min. The pH and oxidation value of milk did not change significanty as a result of the treatments. The β-carotene content was significantly reduced and the total microbial count in milk decreased parallel to the longer exposure time. Also, it was through this longer exposure time by ozone that the L* values of milk increased significantly (p < 0.05), while the b* values significantly decreased. To the best of our knowledge, this is the first study that uses ozone to degrade AFM₁ in milk.     

Immunomodulating Activity of Nymphaea rubra Roxb. Extracts: Activation of Rat Dendritic Cells and Improvement of the TH1 Immune Response

Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 μg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a T_H1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T_H1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs.     

The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches,Interaction with the Long Noncoding HOTAIR RNA

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the ¹H^N-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, ¹H^α-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2^T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR₁₄₀ (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex.     

The Valence Band Structure of the [Ni(Salen)] Complex: An Ultraviolet,Soft X-ray and Resonant Photoemission Spectroscopy Study

The valence band photoemission (VB PE) spectra of the [Ni(Salen)] molecular complex were measured by ultraviolet, soft X-ray and resonant photoemission (ResPE) using photons with energies ranging from 21.2 eV to 860 eV. It was found that the Ni 3d atomic orbitals’ (AOs) contributions are most significant for molecular orbitals (MOs), which are responsible for the low-energy PE band at a binding energy of 3.8 eV in the VB PE spectra. In turn, the PE bands in the binding energies range of 8–16 eV are due to the photoionization of the MOs of the [Ni(Salen)] complex with dominant contributions from C 2p AOs. A detailed consideration was made for the ResPE spectra obtained using photons with absorption resonance energies in the Ni 2p_3/2, N 1s, and O 1s Near-Edge X-ray Absorption Fine Structure (NEXAFS) spectra. A strong increase in the intensity of the PE band ab was found when using photons with an energy 854.4 eV in the Ni 2p_3/2 NEXAFS spectrum. This finding is due to the high probability of the participator-Auger decay of the Ni 2p_3/2⁻¹3d⁹ excitation and confirms the relationship between the PE band ab with the Ni 3d-derived MOs.     

Antiproliferative agents that interfere with the cell cycle at the G1-->S transition: further development and characterization of a small library of stilbene-derived compounds

In this continuation of our research on derivatives containing the stilbene privileged structure or that are derived from it, we report the results of further studies carried out on the previously initiated collection of compounds. We used a parallel synthetic approach to rapidly obtain small sets of compounds and started the annotation of the library in progress by calculating some physicochemical properties to be eventually correlated with biological activities. A pharmacophore for the antiproliferative activity was also built to summarize the features of the library. We evaluated the antiproliferative and pro-apoptotic activities of all compounds as well as the cell-cycle effects of some representative compounds. After in-depth investigations, 3'-phenyl-[1,1';4',1']terphenyl-4,3',5'-triol showed the most interesting biological profile, as it interferes with cell-cycle progression at the G(1)-->S transition, acting on retinoblastoma phosphorylation and inducing cell differentiation.     

Fourier Transform Infrared Microspectroscopy Combined with Principal Component Analysis and Artificial Neural Networks for the Study of the Effect of β-Hydroxy-β-Methylbutyrate (HMB) Supplementation on Articular Cartilage

The potential of Fourier Transform infrared microspectroscopy (FTIR microspectroscopy) and multivariate analyses were applied for the classification of the frequency ranges responsible for the distribution changes of the main components of articular cartilage (AC) that occur during dietary β-hydroxy-β-methyl butyrate (HMB) supplementation. The FTIR imaging analysis of histological AC sections originating from 35-day old male piglets showed the change in the collagen and proteoglycan contents of the HMB-supplemented group compared to the control. The relative amount of collagen content in the superficial zone increased by more than 23% and in the middle zone by about 17%, while no changes in the deep zone were observed compared to the control group. Considering proteoglycans content, a significant increase was registered in the middle and deep zones, respectively; 62% and 52% compared to the control. AFM nanoindentation measurements collected from animals administered with HMB displayed an increase in AC tissue stiffness by detecting a higher value of Young’s modulus in all investigated AC zones. We demonstrated that principal component analysis and artificial neural networks could be trained with spectral information to distinguish AC histological sections and the group under study accurately. This work may support the use and effectiveness of FTIR imaging combined with multivariate analyses as a quantitative alternative to traditional collagenous tissue-related histology.