In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

Exploring potooxidative degradation pathways of harmol and harmalol alkaloids in water: effects of pH, excitation sources and atmospheric conditions.

This work explores the photochemical degradation of cationic species of 7-hydroxy-1-methyl-2H-pyrido[3,4-b]indole or harmol (1C) and the corresponding partially hydrogenated derivative 7-hydroxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole or harmalol (2C) in aqueous solution. UV-visible absorption and fluorescence emission spectroscopy coupled with multivariate data analysis (MCR-ALS and PARAFAC), HPLC and HRESI-MS techniques were used for both quantitative and qualitative analysis. The formation of hydrogen peroxide reactive oxygen species (ROS) was quantified, and the influence of pH, oxygen partial pressure and photoexcitation source on the photochemical degradation of both compounds was assessed. The potential implications on the biosynthesis of ßCs and their biological role in living systems are discussed.

Distribution of photoactive ß-carboline alkaloids across Passiflora caerulea floral organs.

This study reports valuable information regarding the presence and concentration of a series of photoactive ß-carboline (ßCs) alkaloids (norharmane, harmane, harmine, harmol, harmaline, and harmalol) and their distribution across the floral age and organs of Passiflora caerulea. UHPLC-MS/MS data reported herein reveal that the ßCs' content ranged from 1 to 110 µg kg-1 , depending on the floral organ and age. In certain physiologically relevant organs, such as anthers, ßCs' content was one order of magnitude higher than in other organs, suggesting a special role for ßCs in this specific organ. ßCs' content also varied in a structure-dependent manner. Alkaloids bearing a hydroxyl group at position C(7) of the main ßC ring were present at concentrations one order of magnitude higher than other ßC derivatives investigated. UV-visible and fluorescence spectroscopy of the flower extracts provided complementary information regarding other biologically relevant groups of chromophores (phenolic/indolic derivatives, flavonoids/carotenes, and chlorophylls). Since flowers are constantly exposed to solar radiation, the presence of photoactive ßCs in floral organs may have several (photo)biological implications that are further discussed.

Alkylation converts riboflavin into an efficient photosensitizer of phospholipid membranes.

A new decyl chain [-(CH2)9CH3] riboflavin conjugate has been synthesized and investigated. A nucleophilic substitution (SN2) reaction was used for coupling the alkyl chain to riboflavin (Rf), a model natural photosensitizer. As expected, the alkylated compound (decyl-Rf) is significantly more lipophilic than its precursor and efficiently intercalates within phospholipid bilayers, increasing its fluorescence quantum yield. The oxidative damage to lipid membranes photoinduced by decyl-Rf was investigated in large and giant unilamellar vesicles (LUVs and GUVs, respectively) composed of different phospholipids. Using a fluorogenic probe, fast radical formation and singlet oxygen generation was demonstrated upon UVA irradiation in vesicles containing decyl-Rf. Photosensitized formation of conjugated dienes and hydroperoxides, and membrane leakage in LUVs rich in poly-unsaturated fatty acids were also investigated. The overall assessment of the results shows that decyl-Rf is a significantly more efficient photosensitizer of lipids than its unsubstituted precursor and that the association to lipid membranes is key to trigger phospholipid oxidation. Alkylation of hydrophilic photosensitizers as a simple and general synthetic tool to obtain efficient photosensitizers of biomembranes, with potential applications, is discussed.

Synthesis, Characterization and Photocleavage of Bis-decyl Pteroic Acid: A Folate Derivative with Affinity to Biomembranes†.

Here, we provide mechanistic insight to the photocleavage of a compound in the folate family, namely pteroic acid. A bis-decyl chain derivative of pteroic acid was synthesized, structurally characterized and photochemically investigated. We showed that, like folic acid, pteroic acid and the decylated derivative undergo a photocleavage reaction in the presence of H2 O, while no reaction was observed in methanol solution. Furthermore, density functional theory calculations were carried out to predict relative stabilities of hypothetical mono-, bis- and tris-decylated pteroic acid derivatives to help rationalize the regioselectivity of the bis-decyl pteroic acid product. Additionally, the lipophilicity of the bis-decyl pteroic acid appears to confer a hydrophobic property enabling an interaction with biomembranes.

Antibacterial activity against enterovirulent Escherichia coli strains from Bacillus amyloliquefaciens B31 and Bacillus subtilis subsp. subtilis C4: MALDI-TOF MS profiling and MALDI TOF/TOF MS structural analysis on lipopeptides mixtures.

In this work, two Bacillus strains isolated from honey (Bacillus subtilis subsp. subtilis C4; access code HQ828992) and from a waste of an artisanal tannery (Bacillus amyloliquefaciens B31; access code KP893752) were evaluated in order to determine their antibacterial activity against five enteropathogenic Escherichia coli strains. The number of viable cultivable cells of the different strains of E. coli analyzed was determined by plate count. The crude cell-free supernatants of both Bacillus strains exerted anti-E. coli activities, whereas only the lipopeptide fraction of B31 had significant E. coli inhibition. The lipopeptides produced by the Bacillus were analyzed using matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS). The analysis was conducted combining the profiles (fingerprints) of the lipopeptides mixture and the individual lipopeptide fragmentation (tandem mass spectrometry [MS/MS] mode), both obtained from the same lipopeptides mixture sample, for higher output. Data obtained from C4 and B31 revealed that surfactin homologues were the most abundant lipopeptides produced by both strains studied. Additionally, kurstakin, iturin, and fengycin homologues were detected. Using the MS/MS mode, it was demonstrated that isobar compounds belonging to different families were produced by each Bacillus strain (e.g., C-16 bacillomycin D was detected in B31 samples, meanwhile C-15 iturin C was detected in C4). MS/MS analysis contributed with relevant information about the type of lipopeptides synthesized by Bacillus strains studied in this work.

Antibacterial activity of Bacillus licheniformis B6 against viability and biofilm formation of foodborne pathogens of health importance.

We studied a strain of Bacillus isolated from an artisanal tannery in Salta, Argentina. It was identified as Bacillus licheniformis B6 by 16 S phylogenetic analysis and MALDI TOF (GenBank accession code No. KP776730). The synthesis of lipopeptides by B6 and their antibacterial activity against clinical pathogenic strains was analyzed both in the cell-free supernatant (CFS) and in the crude fraction of lipopeptides (LF). Overall, the CFS did not significantly reduce the viability of the studied strains (Staphylococcus aureus 269 and ATCC 43,300, Escherichia coli 4591 and 25,922, Klebsiella sp. 1087 and 1101). However, LF at 9 mg/mL reduced the viability of those pathogenic strains by 2 and 3 log orders compared to those of the control. When the effects of LF and ampicillin were compared, they showed different sensitivity against pathogenic strains. For example, E. coli 4591 was the strain most resistant to ampicillin, requiring 250 mg/mL of antibiotic to achieve the same inhibitory effect as 9 mg/mL of B6 LF. SEM observations of the effect of LF on biofilm formation by E. coli 4591 and Klebsiella sp. 1087 clearly showed that biofilm structures were destabilized, these strains turning into weak biofilm formers. Signals in the CFS and LF corresponding to kurstakin and iturin were identified by MALDI TOF. Interestingly, surfactin was detected, rather than lichenysin, the expected lipopeptide in B. licheniformis species. Signals of bacitracin and fengycins were also found, the latter with a higher number of homologues and relative intensity in the LF than the other lipopeptides. These results show that the lipopeptides synthesized by B. licheniformis B6 have both potential antibacterial and anti-biofilm activity against pathogenic bacteria of health importance.

Dynamics and stabilization mechanism of mitochondrial cristae morphofunction associated with turgor-driven cardiolipin biosynthesis under salt stress conditions.

Maintaining energy production efficiency is of vital importance to plants growing under changing environments. Cardiolipin localized in the inner mitochondrial membrane plays various important roles in mitochondrial function and its activity, although the regulation of mitochondrial morphology to various stress conditions remains obscure, particularly in the context of changes in cellular water relations and metabolisms. By combining single-cell metabolomics with transmission electron microscopy, we have investigated the adaptation mechanism in tomato trichome stalk cells at moderate salt stress to determine the kinetics of cellular parameters and metabolisms. We have found that turgor loss occurred just after the stress conditions, followed by the contrasting volumetric changes in mitochondria and cells, the accumulation of TCA cycle-related metabolites at osmotic adjustment, and a temporal increase in cardiolipin concentration, resulting in a reversible topological modification in the tubulo-vesicular cristae. Because all of these cellular events were dynamically observed in the same single-cells without causing any disturbance for redox states and cytoplasmic streaming, we conclude that turgor pressure might play a regulatory role in the mitochondrial morphological switch throughout the temporal activation of cardiolipin biosynthesis, which sustains mitochondrial respiration and energy conversion even under the salt stress conditions.

Direct evidence for dynamics of cell heterogeneity in watercored apples: turgor-associated metabolic modifications and within-fruit water potential gradient unveiled by single-cell analyses.

Watercore is a physiological disorder in apple (Malus × domestica Borkh.) fruits that appears as water-soaked tissues adjacent to the vascular core, although there is little information on what exactly occurs at cell level in the watercored apples, particularly from the viewpoint of cell water relations. By combining picolitre pressure-probe electrospray-ionization mass spectrometry (picoPPESI-MS) with freezing point osmometry and vapor pressure osmometry, changes in cell water status and metabolisms were spatially assayed in the same fruit. In the watercored fruit, total soluble solid was lower in the watercore region than the normal outer parenchyma region, but there was no spatial difference in the osmotic potentials determined with freezing point osmometry. Importantly, a disagreement between the osmotic potentials determined with two methods has been observed in the watercore region, indicating the presence of significant volatile compounds in the cellular fluids collected. In the watercored fruit, cell turgor varied across flesh, and a steeper water potential gradient has been established from the normal outer parenchyma region to the watercore region, retaining the potential to transport water to the watercore region. Site-specific analysis using picoPPESI-MS revealed that together with a reduction in turgor, remarkable metabolic modifications through fermentation have occurred at the border, inducing greater production of watercore-related volatile compounds, such as alcohols and esters, compared with other regions. Because alcohols including ethanol have low reflection coefficients, it is very likely that these molecules would have rapidly penetrated membranes to accumulate in apoplast to fill. In addition to the water potential gradient detected here, this would physically contribute to the appearance with high tissue transparency and changes in colour differences. Therefore, it is concluded that these spatial changes in cell water relations are closely associated with watercore symptoms as well as with metabolic alterations.

Spectroscopic and quantum chemical characterization of the ground and lowest electronically excited singlet and triplet states of halo- and nitro-harmines in aqueous media.

Halogenated and nitro ß-carboline (ßCs) alkaloids have garnered increasing interest for their role in a broad range of biological, pharmacological and biotechnological processes. Addressing their spectroscopic and photophysical properties provide tools to further explore the presence of these alkaloids in complex biological matrices. In addition, these studies help to elucidate processes where these alkaloids are involved. The UV-visible and steady-state room temperature fluorescence of bromo- and nitro-harmines in an aqueous environment at different pHs, low-temperature phosphorescence (at 77 K) and quantum yields of singlet oxygen production are reported herein. Singlet (S0 and S1) and triplet (T1) electronic states are further analyzed using density functional theory (DFT) and the results compared with experimental data. Data are discussed in the framework of potential biotechnological applications of these ßC alkaloids.

Endosperm cell size reduction caused by osmotic adjustment during nighttime warming in rice.

High night temperature (HNT) often reduces yield in field crops. In rice, HNT during the ripening stage diminishes endosperm cell size, resulting in a considerable reduction in final kernel weight; however, little is known about the underlying mechanisms at cell level. In this study, we performed picolitre pressure-probe-electrospray-ionization mass spectrometry to directly determine metabolites in growing inner endosperm cells of intact seeds produced under HNT conditions, combining with 13C feeding and water status measurements including in situ turgor assay. Microscopic observation in the inner zone suggested that approximately 24.2% of decrease in cell expansion rate occurred under HNT at early ripening stage, leading to a reduction in cell volume. It has been shown that HNT-treated plants were subjected to mild shoot water deficit at night and endosperm cell turgor was sustained by a decline in osmotic potential. Cell metabolomics also suggests that active solute accumulation was caused by a partial inhibition of wall and starch biosynthesis under HNT conditions. Because metabolites were detected in the single cells, it is concluded that a partial arrest of cell expansion observed in the inner endosperms was caused by osmotic adjustment at mild water deficit during HNT conditions.

Photophysical and spectroscopic features of 3,4-dihydro-ß-carbolines: a combined experimental and theoretical approach.

3,4-Dihydro-ß-carbolines (DHßCs) are a set of endogenously synthesized alkaloids spread over a great variety of living species (e.g., plants, animals and microorganisms), playing a broad spectrum of biological, biochemical and/or pharmacological roles, in a structure-dependent manner. Addressing unresolved fundamental aspects related to the photophysical properties of DHßCs might help to gain further insights into the molecular basis of the mechanisms of the biological processes where these alkaloids are involved. In this work, the UV-visible spectroscopic features of DHßCs are revisited and they are further analyzed by calculations at the Density Functional Theory (DFT) level. In addition, steady-state and time-resolved fluorescence spectroscopy, as well as quantitative singlet oxygen production analysis is reported. Data obtained herein are discussed in the framework of the potential biological role of these alkaloids.

Design and Effects of the Cinnamic Acids Chemical Structures as Organocatalyst on the Polymerization of Benzoxazines.

This study focuses on the catalytic effect of the two geometric isomers of a cinnamic acid derivative, E and Z-forms of 3-methoxycinnamic acid (3OMeCA), analyzing the influence of their chemical structures. E and Z-3OMeCA isomers show very good catalytic effect in the polymerization of benzoxazines, decreasing by 40 and 55 °C, respectively, the polymerization temperatures, for catalyst contents of up to 10% w/w. Isothermal polymerizations show that polymerizations are easily realized and analyzed at temperatures as low as 130 °C and at much shorter times using Z-3OMeCA instead of E-3OMeCA. Thus, both cinnamic acids are good catalysts, with Z-3OMeCA being better. The molecular reasons for this difference and mechanistic implications in benzoxazine polymerizations are also presented.

Substituent Effects on NMR Spectroscopy of 2,2-Dimethylchroman-4-one Derivatives: Experimental and Theoretical Studies.

The attribution of 1H and 13C NMR signals of a library of 5-, 6- and 7-substituted 2,2-dimethylchroman-4-one derivatives is reported. Substituent effects were interpreted in terms of the Hammett equation, showing a good correlation for carbons para- to the substituent group, not for the meta- ones. Similarly, the Lynch correlation shows the additivity of the substituent chemical shifts in the case of both H and C nuclei, again with the exception of the carbons in the meta- position. Density Functional Theory (DFT)-predicted 1H and 13C chemical shifts correspond closely with experimentally observed values, with some exceptions for C NMR data; however, the correlation is valid only for the aromatic moiety and cannot be extended to the heterocyclic ring of the chroman-4-one scaffold.

Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin.

Whey protein is one of the most relevant co-products manufactured by the dairy industry and it is a powerful environmental pollutant. Therefore, the enzymatic hydrolysis of whey protein concentrate (WPC 35) to produce antioxidant peptides is an innovative approach which can provide added value to whey. The WPC 35 hydrolysis with trypsin was carried out for 4.31 h at 41.1 °C with an enzyme/substrate ratio of 0.017. Under such hydrolysis conditions, the peptides produced have the highest radical scavenging activity and cytoprotector effect. The WPC hydrolysate and a permeate ≤3 kDa were characterized by SDS-page, RP-HPLC and MALDI-TOF-MS. Furthermore, O2•- and HO• scavenging activity and the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18) were determined. In this study, strong antioxidant and cytoprotective peptides were obtained from a low-cost dairy industry product, which could improve consumers' health when used as functional ingredients.

Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose.

Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture. •Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.

On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice.

Although a loss of healthy pollen grains induced by metabolic heat responses has been indicated to be a major cause of heat-induced spikelet sterility under global climate change, to date detailed information at pollen level has been lacking due to the technical limitations. In this study, we used picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS) to directly determine the metabolites in heat-treated single mature pollen grains in two cultivars, heat-tolerant cultivar, N22 and heat-sensitive cultivar, Koshihikari. Heat-induced spikelet fertility in N22 and Koshihikari was 90.0% and 46.8%, respectively. While no treatment difference in in vitro pollen viability was observed in each cultivar, contrasting varietal differences in phosphatidylinositol (PI)(34:3) have been detected in mature pollen, together with other 106 metabolites. Greater PI content was detected in N22 pollen regardless of the treatment, but not for Koshihikari pollen. In contrast, there was little detection for phosphoinositide in the single mature pollen grains in both cultivars. Our findings indicate that picoPPESI-MS analysis can efficiently identify the metabolites in intact single pollen. Since PI is a precursor of phosphoinositide that induces multiple signaling for pollen germination and tube growth, the active synthesis of PI(34:3) prior to germination may be closely associated with sustaining spikelet fertility even at high temperatures.

Understanding the role played by protic ionic liquids (PILs) and the substituent effect for enhancing the generation of Z-cinnamic acid derivatives†.

Photoisomerization of a series of substituted E-cinnamic acids in MeCN in their acid forms and as their corresponding protic ionic liquids (PILs) with light of 300 nm is studied. The nature, strength, number, and position effects of substituents on the photochemical behavior of E-cinnamic derivatives are investigated. The photosensitization of the reaction in the presence of Michler's ketone is also studied at 366 nm and it demonstrates that the triplet-excited state is involved in the reaction. As the presence of n-butylamine needed to form the PILs significantly increases the photoproduct yields in all cases, the role of the PILs is also discussed. Thus, understanding of these fundamental aspects has allowed us to establish an excellent and practical synthetic protocol for successfully synthesizing Z-cinnamic acids.

A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification.

Bevacizumab is a vascular endothelial growth factor (VEGF)-directed monoclonal antibody (mAb) used for the treatment of several human cancers. Given that bevacizumab is administered intravenously, it must have extremely high purity, which is achieved by purification with protein A affinity chromatography (AC). However, protein A is a very expensive ligand, thereby increasing the cost of purification. Furthermore, the harsh elution conditions required to recover bevacizumab from the AC column can damage both the mAb and protein A. In contrast, short peptides show higher stability, easier synthesis and lower cost and are therefore ideal ligands for AC. In the present study, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment that binds bevacizumab, was synthesized and immobilized on agarose. The peptidyl-agarose showed affinity for bevacizumab, with an equilibrium dissociation constant value of 2.2±0.5 x 10-7 M under optimal conditions. Samples of CHO cell filtrate producing bevacizumab were loaded on the peptidyl-agarose chromatography column. Bevacizumab was recovered from the elution fraction with a yield of 94% and a purity of 98%. The maximum capacity (qm) 38±2 mg of bevacizumab per mL of matrix was comparable to that of commercial protein A matrices. Moreover, the peptide ligand showed greater stability and a lower cost than protein A. Unlike peptides previously reported for IgG purification, the ligand described herein allows mAb elution under mild conditions, thereby favoring the integrity of bevacizumab. The lack of Trp, Met or Cys in the peptide prevents its oxidation and extends the useful life of the chromatographic matrix.

Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer.

The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). Particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV-visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography, and mass spectrometry is used to establish the Ub changes triggered by UV-A irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes, that is, formation of a Ptr-Ub adduct, dimerization, and fragmentation of the protein.