Identification and Characterization of Neuroprotective Properties of Thaumatin-like Protein 1a from Annurca Apple Flesh Polyphenol Extract.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial neurodegenerative disorders that are mostly treated with drugs inhibiting key enzymes of cholinergic and aminergic neurotransmission, such as acetyl and butyryl cholinesterase (AChE, BuChE) or monoamine oxidases (MAO)-A/B, and of Aß1-40 aggregation. Diet plant components with multitarget functions are promising compounds in the prevention of AD and PD. Our aim was to identify neuroprotective compounds from Annurca apple polyphenol extract (AFPE). METHODS: AFPE was fractionated by gel filtration, and the eluted peaks were subjected to chemical analyses (i.e., RP-HPLC and mass spectrometry), determination of inhibitory enzyme activity and cell effects by MTT, and morphology assays. RESULTS: In AFPE, we identified thaumatin-like protein 1a, belonging to the pathogenesis-related protein (PR) family. This protein showed the best inhibitory activity on AChE, MAO-A (IC50 = 5.53 µM and 1.71 µM, respectively), and Aß1-40 fibril aggregation (IC50 = 9.16 µM), compared to AFPE and other polyphenol-containing fractions. Among the latter, Peak 4 reverted Aß fibril formation (IC50 = 104.87 µM). Moreover, thaumatin-like protein 1a protected AGS and MKN-28 cells from serum-deprivation-induced stress conditions. CONCLUSIONS: We showed that AFPE exerted neuroprotective functions not only through its polyphenols but also through thaumatin-like protein 1a, which acted like a multitarget molecule.

Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids.

Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents' brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

A robust nanoLC high-resolution mass spectrometry methodology for the comprehensive profiling of lactic acid bacteria in milk kefir.

Consumer attention to functional foods containing probiotics is growing because of their positive effects on human health. Kefir is a fermented milk beverage produced by bacteria and yeasts. Given the emerging kefir market, there is an increasing demand for new methodologies to certify product claims such as colony-forming units/g and bacterial taxa. MALDI-TOF MS proved to be useful for the detection/identification of bacteria in clinical diagnostics and agri-food applications. Recently, LC-MS/MS approaches have also been applied to the identification of proteins and proteotypic peptides of lactic acid bacteria in fermented food matrices. Here, we developed an innovative nanoLC-ESI-MS/MS-based methodology for profiling lactic acid bacteria in commercial and artisanal milk kefir products as well as in kefir grains at the genus, species and subspecies level. The proposed workflow enables the authentication of kefir label claims declaring the presence of probiotic starters. An overview of the composition of lactic acid bacteria was also obtained for unlabelled kefir highlighting, for the first time, the great potential of LC-MS/MS as a sensitive tool to assess the authenticity of fermented foods.

MucR from Sinorhizobium meliloti: New Insights into Its DNA Targets and Its Ability to Oligomerize.

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.

Phytochemical Investigation of Myrcianthes cisplatensis: Structural Characterization of New p-Coumaroyl Alkylphloroglucinols and Antimicrobial Evaluation against Staphylococcus aureus.

Species of Myrtaceae Juss., the ninth largest family of flowering plants, are a valuable source of bioactive specialized metabolites. A leading position belongs to phloroglucinol derivatives, thanks to their unusual structural features and biological and pharmacological properties. Myrcianthes cisplatensis (Cambess.) O. Berg, a common tree on the banks of rivers and streams of Uruguay, southern Brazil, and northern Argentina, with aromatic leaves, is known as a diuretic, febrifuge, tonic, and good remedy for lung and bronchial diseases. Despite knowledge about traditional use, few data on its phytochemical properties have been reported in the literature. The methanol extract of M. cisplatensis, grown in Arizona, USA, was first partitioned between dichloromethane and water and then with ethyl acetate. The enriched fractions were evaluated using a broth microdilution assay against Staphylococcus aureus ATCC 29213 and 43300 (methicillin-resistant S. aureus (MRSA)). The potential antimicrobial activity seemed to increase in the dichloromethane extract, with a MIC value of 16 µg/mL against both strains. Following a bio-guided approach, chromatographic techniques allowed for isolating three coumarin derivatives, namely endoperoxide G3, catechin, and quercitrin, and four new p-coumaroyl alkylphloroglucinol glucosides, named p-coumaroylmyrciacommulone A-D. Their structures were characterized through spectroscopic techniques: 2D-NMR experiments (HSQC, HMBC, and HSQC-TOCSY) and spectrometric analyses (HR-MS). The antimicrobial assessment of pure compounds against S. aureus ATCC 29213 and ATCC 43300 demonstrated the best activity for p-coumaroylmyrciacommulone C and D with the growth inhibition of 50% at 32 µg/mL against both strains of S. aureus.

Structural and functional characterization of the cytotoxic protein ledodin, an atypical ribosome-inactivating protein from shiitake mushroom (Lentinula edodes).

We have purified ledodin, a cytotoxic 22-kDa protein from shiitake mushroom (Lentinula edodes) consisting of a 197 amino acid chain. Ledodin possessed N-glycosylase activity on the sarcin-ricin loop of mammalian 28S rRNA and inhibited protein synthesis. However, it was not active against insect, fungal, and bacterial ribosomes. In vitro and in silico studies suggested that ledodin exhibits a catalytic mechanism like that of DNA glycosylases and plant ribosome-inactivating proteins. Moreover, the sequence and structure of ledodin was not related to any protein of known function, although ledodin-homologous sequences were found in the genome of several species of fungi, some edible, belonging to different orders of the class Agaricomycetes. Therefore, ledodin could be the first of a new family of enzymes widely distributed among this class of basidiomycetes. The interest of these proteins lies both, in the fact that they can be a toxic agent of some edible mushrooms and in their application in medicine and biotechnology.

Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS).

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

Nusinersen mitigates neuroinflammation in severe spinal muscular atrophy patients.

BACKGROUND: Neuroinflammation contributes to the onset and progression of neurodegenerative diseases, but has not been specifically investigated in patients affected by severe and milder forms of spinal muscular atrophy (SMA). METHODS: In this two-center retrospective study, we investigated signatures of neuroinflammation in forty-eight pediatric male and female SMA1 (n = 18), male and female SMA2 (n = 19), and female SMA3 (n = 11) patients, as well as in a limited number of male and female non-neurological control subjects (n = 4). We employed a Bio-Plex multiplex system based on xMAP technology and performed targeted quantitative analysis of a wide range of pro- and anti-inflammatory cytokines (chemokines, interferons, interleukins, lymphokines and tumor necrosis factors) and neurotrophic factors in the cerebrospinal fluid (CSF) of the study cohort before and after Nusinersen treatment at loading and maintenance stages. RESULTS: We find a significant increase in the levels of several pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-2, IL-8, IL-12, IL-17, MIP-1α, MCP-1, and Eotaxin) and neurotrophic factors (PDGF-BB and VEGF) in the CSF of SMA1 patients relative to SMA2 and SMA3 individuals, who display levels in the range of controls. We also find that treatment with Nusinersen significantly reduces the CSF levels of some but not all of these neuroinflammatory molecules in SMA1 patients. Conversely, Nusinersen increases the CSF levels of proinflammatory G-CSF, IL-8, MCP-1, MIP-1α, and MIP-1ß in SMA2 patients and decreases those of anti-inflammatory IL-1ra in SMA3 patients. CONCLUSIONS: These findings highlight signatures of neuroinflammation that are specifically associated with severe SMA and the neuro-immunomodulatory effects of Nusinersen therapy.

Spinal muscular atrophy (SMA) is an inherited disorder which leads to muscle weakening. Three therapies have recently been developed, including Nusinersen. However, the effect of SMA on the immune system and how this could be affected by Nusinersen is unknown. The immune system protects the body from infection and, in some disorders, misfunctions and damages the body in the absence of infection. Here, we analyze components of the immune system in body fluids from SMA patients before and after treatment with Nusinersen. The immune system was found to be more active in patients with more severe disease. Treatment with Nusinersen reduced the levels of some, but not all of these, components of the immune system. Thus, treatments that impact the immune system might improve symptoms in patients with SMA.

Molecular Fingerprint of Human Pathological Synoviocytes in Response to Extractive Sulfated and Biofermentative Unsulfated Chondroitins.

Pharma-grade extractive chondroitin sulfate (CS) is widely used for osteoarthritis (OA) treatment. Recently, unsulfated biofermentative chondroitin (BC) proved positive effects in OA in vitro model. This study, based on primary pathological human synoviocytes, aimed to analyze, by a multiplex assay, a panel of OA-related biomarkers in response to short-term treatments with bovine (CSb), pig (CSp) and fish (CSf) chondroitins, in comparison to BC. As expected, all samples had anti-inflammatory properties, however CSb, CSf and especially BC affected more cytokines and chemokines. Based on these results and molecular weight similarity, CSf and BC were selected to further explore the synoviocytes' response. In fact, Western blot analyses showed CSf and BC were comparable, downregulating OA-related biomarkers such as the proteins mTOR, NF-kB, PTX-3 and COMP-2. Proteomic analyses, performed by applying a nano-LC-MS/MS TMT isobaric labelling-based approach, displayed the modulation of both common and distinct molecules to chondroitin treatments. Thus, CSf and BC modulated the biological mediators involved in the inflammation cascade, matrix degradation/remodeling, glycosaminoglycans' synthesis and cellular homeostasis. This study helps in shedding light on different molecular mechanisms related to OA disease that may be potentially affected not only by animal-source chondroitin sulfate but also by unsulfated biofermentative chondroitin.

Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex.

BACKGROUND: Imprinting Control Regions (ICRs) are CpG-rich sequences acquiring differential methylation in the female and male germline and maintaining it in a parental origin-specific manner in somatic cells. Despite their expected high mutation rate due to spontaneous deamination of methylated cytosines, ICRs show conservation of CpG-richness and CpG-containing transcription factor binding sites in mammalian species. However, little is known about the mechanisms contributing to the maintenance of a high density of methyl CpGs at these loci. RESULTS: To gain functional insights into the mechanisms for maintaining CpG methylation, we sought to identify the proteins binding the methylated allele of the ICRs by determining the interactors of ZFP57 that recognizes a methylated hexanucleotide motif of these DNA regions in mouse ESCs. By using a tagged approach coupled to LC-MS/MS analysis, we identified several proteins, including factors involved in mRNA processing/splicing, chromosome organization, transcription and DNA repair processes. The presence of the post-replicative mismatch-repair (MMR) complex components MSH2 and MSH6 among the identified ZFP57 interactors prompted us to investigate their DNA binding profile by chromatin immunoprecipitation and sequencing. We demonstrated that MSH2 was enriched at gene promoters overlapping unmethylated CpG islands and at repeats. We also found that both MSH2 and MSH6 interacted with the methylated allele of the ICRs, where their binding to DNA was mediated by the ZFP57/KAP1 complex. CONCLUSIONS: Our findings show that the MMR complex is concentrated on gene promoters and repeats in mouse ESCs, suggesting that maintaining the integrity of these regions is a primary function of highly proliferating cells. Furthermore, the demonstration that MSH2/MSH6 are recruited to the methylated allele of the ICRs through interaction with ZFP57/KAP1 suggests a role of the MMR complex in the maintenance of the integrity of these regulatory regions and evolution of genomic imprinting in mammalian species.

Tailoring the Structure of Cell Penetrating DNA and RNA Binding Nucleopeptides.

Synthetic nucleic acid interactors represent an exciting research field due to their biotechnological and potential therapeutic applications. The translation of these molecules into drugs is a long and difficult process that justifies the continuous research of new chemotypes endowed with favorable binding, pharmacokinetic and pharmacodynamic properties. In this scenario, we describe the synthesis of two sets of homo-thymine nucleopeptides, in which nucleobases are inserted in a peptide structure, to investigate the role of the underivatized amino acid residue and the distance of the nucleobase from the peptide backbone on the nucleic acid recognition process. It is worth noting that the CD spectroscopy investigation showed that two of the reported nucleopeptides, consisting of alternation of thymine functionalized L-Orn and L-Dab and L-Arg as underivatized amino acids, were able to efficiently bind DNA and RNA targets and cross both cell and nuclear membranes.

Myoglobin from Atlantic and Tinker mackerels: Purification, characterization and its possible use as a molecular marker.

Here, we report the characterization (purification, autoxidation rate, pseudoperoxidase activity) and amino acid sequence determination of S. scombrus (Atlantic mackerel) and S. colias (Tinker mackerel) mioglobins (Mbs), considering the increasing consumption of fresh and canned mackerel meat and Mb implication in meat storage (e.g.: browning and lipid oxidation). We found that Atlantic mackerel Mb has major autoxidation rate (0.204 ± 0.013 h-1) compared to Tinker mackerel Mb (0.140 ± 0.009 h-1), while the pseudoperoxidase activity is major for Tinker mackerel (Km: 87.71 ± 7.19 µM; kcat: 0.32 s-1) Mb with respect to Atlantic mackerel (Km: 96.08 ± 6.91 µM; kcat: 0.50 s-1). These functional differences are confirmed by primary structure determination, in which six amino acid substitutions are found, with the first N-terminal amino acid residue acetylated. Furthermore, we predicted by AphaFold 3D model both fish Mbs and used them to investigate the possible structural differences. In addition, phylogenetic analysis using Mb sequences from Scombridae family confirms that Atlantic and Tinker mackerels are two distinct species. Finally, an analytic qualitative RP-HPLC method to distinguish S. scombrus and S. colias specimens was developed considering the different retention times of the two mackerel apoMbs.

Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond.

Cannabidiolic acid (CBDA) is the main precannabinoid in industrial hemp. It represents a common constituent of hemp seed oil, but mainly abundant in the aerial parts of the plant (including their processing waste). Thus, the optimization of fast and low-cost purification strategies is mandatory, as well as a deep investigation on its nutraceutical and cosmeceutical properties. To this purpose, CBDA content in hemp seed oil is evaluated, and its recovery from wasted leaves is favorably achieved. The cytotoxicity screening towards HaCaT cells, by means of MTT, SRB and LDH release assays, suggested it was not able to decrease cell viability or perturb cell integrity up to 10 µM concentration. Thus, the ability of CBDA to differentially modulate the release of proinflammatory cytokines and chemokines mediators has been evaluated, finding that CBDA decreased IFN-γ, CXCL8, CXCL10, CCL2, CCL4 and CCL5, mostly in a dose-dependent manner, with 10 µM tested concentration exerting the highest activity. These data, together with those from assessing antimicrobial activity against Gram(+) and Gram(-) bacteria and the antibiofilm formation, suggest that CBDA is able to counteract the inflammatory response, also preventing bacteria colonization.

Deciphering Molecular Determinants Underlying Penicillium digitatum's Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS.

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum's response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum's response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

The Structural Characterization and Antipathogenic Activities of Quinoin, a Type 1 Ribosome-Inactivating Protein from Quinoa Seeds.

Quinoin is a type 1 ribosome-inactivating protein (RIP) we previously isolated from the seeds of pseudocereal quinoa (Chenopodium quinoa) and is known as a functional food for its beneficial effects on human health. As the presence of RIPs in edible plants could be potentially risky, here we further characterised biochemically the protein (complete amino acid sequence, homologies/differences with other RIPs and three-dimensional homology modeling) and explored its possible defensive role against pathogens. Quinoin consists of 254 amino acid residues, without cysteinyl residues. As demonstrated by similarities and homology modeling, quinoin preserves the amino acid residues of the active site (Tyr75, Tyr122, Glu177, Arg180, Phe181 and Trp206; quinoin numbering) and the RIP-fold characteristic of RIPs. The polypeptide chain of quinoin contains two N-glycosylation sites at Asn115 and Asp231, the second of which appears to be linked to sugars. Moreover, by comparative MALDI-TOF tryptic peptide mapping, two differently glycosylated forms of quinoin, named pre-quinoin-1 and pre-quinoin-2 (~0.11 mg/100 g and ~0.85 mg/100 g of seeds, respectively) were characterised. Finally, quinoin possesses: (i) strong antiviral activity, both in vitro and in vivo towards Tobacco Necrosis Virus (TNV); (ii) a growth inhibition effect on the bacterial pathogens of plants; and (iii) a slight antifungal effect against two Cryphonectria parasitica strains.

Chemical Characterization and Anti-HIV-1 Activity Assessment of Iridoids and Flavonols from Scrophularia trifoliata.

Plants are the everlasting source of a wide spectrum of specialized metabolites, characterized by wide variability in term of chemical structures and different biological properties such antiviral activity. In the search for novel antiviral agents against Human Immunodeficiency Virus type 1 (HIV-1) from plants, the phytochemical investigation of Scrophularia trifoliata L. led us to isolate and characterize four flavonols glycosides along with nine iridoid glycosides, two of them, 5 and 13, described for the first time. In the present study, we investigated, for the first time, the contents of a methanol extract of S. trifoliata leaves, in order to explore the potential antiviral activity against HIV-1. The antiviral activity was evaluated in biochemical assays for the inhibition of HIV-1Reverse Transcriptase (RT)-associated Ribonuclease H (RNase H) activity and HIV-1 Integrase (IN). Three isolated flavonoids, rutin, kaempferol-7-O-rhamnosyl-3-O-glucopyranoside, and kaempferol-3-O-glucopyranoside, 8-10, inhibited specifically the HIV-1 IN activity at submicromolar concentration, with the latter being the most potent, showing an IC50 value of 24 nM.