Part I: surface-enhanced Raman spectroscopy investigation of amino acids and their homodipeptides adsorbed on colloidal silver

Surface-enhanced Raman scattering spectra (SERS) were measured for various amino acids: L-methionine (Met), L-cysteine (Cys), Lglycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, LeuLeu, Phe-Phe, and Pro-Pro) in silver colloidal solutions. The geometry and orientation of the amino acids or dipeptides on the silver surface, and their specific interaction with the surface, were deducted by detailed spectral analysis of the SERS spectra. This analysis has allowed us to propose the particular surface geometry of amino acids or dipeptides and also implied that C-C bonds were almost parallel to the surface, as evidenced by the absence of marker bands in the skeletal C-C stretching region of the spectra. Additionally, using "time-dependent" SERS measurements we solved an existing controversy regarding the binding specificity of Gly-Gly on the silver surface.     

Part III: Surface-enhanced Raman scattering of amino acids and their homodipeptide monolayers deposited onto colloidal gold surface

Surface-enhanced Raman scattering (SERS) spectra were measured for monolayers of various amino acids: L-methionine (Met), L-cysteine (Cys), L-glycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, Leu-Leu, Phe-Phe, and Pro-Pro) deposited onto a colloidal gold surface. Orientation of amino acids and their homodipeptides, as well as specific-competitive interactions of their functional groups with the gold surface, were predicted by detailed spectral analysis of the obtained SERS spectra. The analysis performed allowed us to propose a particular surface geometry for each amino acid and homodipeptide on the gold surface. In addition, we compared the structures of these molecules adsorbed on colloidal gold and silver surfaces.     

Effects of Trichoderma harzianum on Photosynthetic Characteristics and Fruit Quality of Tomato Plants

The beneficial role of fungi from the Trichoderma genus and its secondary metabolites in promoting plant growth, uptake and use efficiency of macronutrients and oligo/micro-nutrients, activation of plant secondary metabolism and plant protection from diseases makes it interesting for application in environmentally friendly agriculture. However, the literature data on the effect of Trichoderma inoculation on tomato fruit quality is scarce. Commercially used tomato cultivars were chosen in combination with indigenous Trichodrema species previously characterized on molecular and biochemical level, to investigate the effect of Trichoderma on photosynthetic characteristics and fruit quality of plants grown in organic system of production. Examined cultivars differed in the majority of examined parameters. Response of cultivar Gružanski zlatni to Trichoderma application was more significant. As a consequence of increased epidermal flavonols and decreased chlorophyll, the nitrogen balance index in leaves has decreased, indicating a shift from primary to secondary metabolism. The quality of its fruit was altered in the sense of increased total flavonoids content, decreased starch, increased Bioaccumulation Index (BI) for Fe and Cr, and decreased BI for heavy metals Ni and Pb. Higher expression of swolenin gene in tomato roots of more responsive tomato cultivar indicates better root colonization, which correlates with observed positive effects of Trichodrema.     

Fungal Biotransformation of 2′-Methylflavanone and 2′-Methylflavone as a Method to Obtain Glycosylated Derivatives

Methylated flavonoids are promising pharmaceutical agents due to their improved metabolic stability and increased activity compared to unmethylated forms. The biotransformation in cultures of entomopathogenic filamentous fungi is a valuable method to obtain glycosylated flavones and flavanones with increased aqueous solubility and bioavailability. In the present study, we combined chemical synthesis and biotransformation to obtain methylated and glycosylated flavonoid derivatives. In the first step, we synthesized 2′-methylflavanone and 2′-methylflavone. Afterwards, both compounds were biotransformed in the cultures of two strains of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5 and Isaria fumosorosea KCH J2. We determined the structures of biotransformation products based on NMR spectroscopy. Biotransformations of 2′-methyflavanone in the culture of B. bassiana KCH J1.5 resulted in three glycosylated flavanones: 2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, 3′-hydroxy-2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, and 2-(2′-methylphenyl)-chromane 4-O-β-d-(4″-O-methyl)-glucopyranoside, whereas in the culture of I. fumosorosea KCH J2, two other products were obtained: 2′-methylflavanone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2-methylbenzoic acid 4-O-β-d-(4′-O-methyl)-glucopyranoside. 2′-Methylflavone was effectively biotransformed only by I. fumosorosea KCH J2 into three derivatives: 2′-methylflavone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-methylflavone 4′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′-methylflavone 5′-O-β-d-(4″-O-methyl)-glucopyranoside. All obtained glycosylated flavonoids have not been described in the literature until now and need further research on their biological activity and pharmacological efficacy as potential drugs.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.     

Alleviation of Osmotic Stress Effects by Exogenous Application of Salicylic or Abscisic Acid on Wheat Seedlings

The aim of the study was to assess the role of salicylic acid (SA) and abscisic acid (ABA) in osmotic stress tolerance of wheat seedlings. This was accomplished by determining the impact of the acids applied exogenously on seedlings grown under osmotic stress in hydroponics. The investigation was unique in its comprehensiveness, examining changes under osmotic stress and other conditions, and testing a number of parameters simultaneously. In both drought susceptible (SQ1) and drought resistant (CS) wheat cultivars, significant physiological and biochemical changes were observed upon the addition of SA (0.05 mM) or ABA (0.1 μM) to solutions containing half-strength Hoagland medium and PEG 6000 (−0.75 MPa). The most noticeable result of supplementing SA or ABA to the medium (PEG + SA and PEG + ABA) was a decrease in the length of leaves and roots in both cultivars. While PEG treatment reduced gas exchange parameters, chlorophyll content in CS, and osmotic potential, and conversely, increased lipid peroxidation, soluble carbohydrates in SQ1, proline content in both cultivars and total antioxidants activity in SQ1, PEG + SA or PEG + ABA did not change the values of these parameters. Furthermore, PEG caused a two-fold increase of endogenous ABA content in SQ1 and a four-fold increase in CS. PEG + ABA increased endogenous ABA only in SQ1, whereas PEG + SA caused a greater increase of ABA content in both cultivars compared to PEG. In PEG-treated plants growing until the harvest, a greater decrease of yield components was observed in SQ1 than in CS. PEG + SA, and particularly PEG + ABA, caused a greater increase of these yield parameters in CS compared to SQ1. In conclusion, SA and ABA ameliorate, particularly in the tolerant wheat cultivar, the harmful effects and after effects of osmotic stress induced by PEG in hydroponics through better osmotic adjustment achieved by an increase in proline and carbohydrate content as well as by an increase in antioxidant activity.     

IFNβ-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages

IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNβ expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating CXCL10 expression in response to IFNβ stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNβ as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNβ-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with CXCL10 promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNβ-induced response in BMDM and THP-1 cell lines.