New insights into the operative network of FaEO,an enone oxidoreductase from <Emphasis Type="Italic">Fragaria x ananassa</Emphasis> Duch.

The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.     

Breast cancer stem cells: The role of sex steroid receptors

Breast cancer(BC) is the most common cancer among women, and current available therapies often have high success rates. Nevertheless, BC might acquire drug resistance and sometimes relapse. Current knowledge about the most aggressive forms of BC points to the role of specific cells with stem properties located within BC, the so-called "BC stem cells"(BCSCs). The role of BCSCs in cancer formation, growth, invasiveness, therapy resistance and tumor recurrence is becoming increasingly clear. The growth and metastatic properties of BCSCs are regulated by different pathways, which are only partially known. Sex steroid receptors(SSRs), which are involved in BC etiology and progression, promote BCSC proliferation, dedifferentiation and migration. However, in the literature,there is incomplete information about their roles. Particularly, there are contrasting conclusions about the expression and role of the classical BC hormonal biomarkers, such as estrogen receptor alpha(ERα), together with scant,albeit promising information concerning ER beta(ERβ) and androgen receptor(AR) properties that control different transduction pathways in BCSCs. In this review, we will discuss the role that SRs expressed in BCSCs play to BC progression and recurrence and how these findings have opened new therapeutic possibilities.     

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

Background

Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic.

Methods

RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes.

Results

Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication.

Conclusions

Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

     

Nitrated nucleosome levels and neuropsychiatric events in systemic lupus erythematosus; a multi-center retrospective case-control study

Background

In patients with systemic lupus erythematosus (SLE) there is no serological test that will reliably distinguish neuropsychiatric (NP) events due to active SLE from those due to other causes. Previously we showed that serum levels of nitrated nucleosomes (NN) were elevated in a small number of patients with NPSLE. Here we measured serum NN in samples from a larger population of patients with SLE and NP events to see whether elevated serum NN could be a marker for NPSLE.

Methods

We obtained serum samples from patients in the Systemic Lupus International Collaborative Clinics (SLICC) inception cohort. This included 216 patients with NP events and two matched controls with SLE but no NP events for each of these patients. For the NP patients we tested samples taken before, during and after the NP event.

Results

Twenty-six patients had events attributed to SLE according to the most stringent SLICC attribution rule. In these patients there was no association between onset of event and elevated serum NN. In 190 patients in whom events were not attributed to SLE by the SLICC rules, median serum NN was elevated at the onset of event (P?=?0.006). The predominant clinical features in this group of 190 patients were headache, mood disorders and anxiety.

Conclusions

Serum NN levels rise at the time of an NP event in a proportion of patients with SLE. Further studies are needed to determine the value of serum NN as a biomarker for NPSLE.

     

Best practice of identification and proteomic analysis of extracellular vesicles in human health and disease

Introduction: Extracellular vesicles are emerging sources of biomarkers for modern preventive and precision medicine. Extracellular vesicles in body fluids offer a unique opportunity for integrative biomarker approaches due to their complex biocargo that includes proteins, lipids, nucleic acids and metabolites. Mass spectrometry-based proteomics data suggest that a significant portion of human proteins are sorted into extracellular vesicles and amenable for biomarker discovery schemes.

Areas covered: this review focuses on key aspects of isolation, quality control and subsequent analysis of blood plasma- and conditioned medium-derived extracellular vesicle proteins, and summarizes the current state-of-the-art in the field. Furthermore, it provides introduction and guidelines for mass spectrometry-based proteomic analysis of extracellular vesicles.

Expert commentary: Comparison of newly developed isolation and purification techniques with classical ultracentrifugation-based approaches are highly recommended. It is also essential to use multiple analytical approaches to characterize the isolated extracellular vesicles prior to characterization of their biocargo. Rigor in data reproducibility, critical data analysis, awareness of potential pitfalls, standardization and benchmarking are required for extracellular vesicle research to fulfil the current expectation that these subcellular structures can become a valid source of next generation biomarkers.     

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC₅₀ 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC₅₀ 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

     

Vessel occlusion in three cultivars of Olea europaea naturally exposed to Xylella fastidiosa in open field

Xylella fastidiosa is a Gram‐negative, xylem‐limited, bacterium which is responsible, in Italy, for the olive quick decline syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In this study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analysing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars “Ogliarola salentina” and “Cellina di Nardò,” and the tolerant cultivar “Leccino.” Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.     

The influence of the nitrogen substitution in three cytisine derivatives as ligands for the neuronal nAChRs: a structural and theoretical study

Three cytisine derivatives, (-)-(7R,9S)-1-phenyl-3-(cytisin-12-yl)propan-1-one (1), (-)-(7R,9S)-1-phenyl-2-(cytisin-12-yl)ethane (2), and (-)-(7R,9S)-1,2-bis(cytisin-12-yl)ethane (3), with different electronic and steric features have been characterized by X-ray analysis and theoretical calculations in order to evaluate how structural modulations affect the intrinsic binding affinity at the neuronal nicotinic receptors (nAChRs). The crystal structures of 1 and 2, which display comparable affinities, are characterized by the same conformation of the cytisine moiety with different orientations of the substituent at N2. In 3, two independent molecules have the pyridinone rings diversely oriented. This compound has a lower affinity with respect to 1 and 2, but it increases the expression of neuronal nAChRs. Compounds 1, 2, and 3 retain the key prerequisite of the classical pharmacophoric models, with sp(3)-N-atom--HBA distances close to the expected value, both in solid state and in solution (theoretical calculations), where, in contrast with the extended in the crystal state, a curled-up conformation has been found, though maintaining the N-substituent in equatorial position.     

Taste-active compounds in a traditional Italian food: 'lampascioni'

Nature is a rich source of taste-active compounds, in particular of plant origin, many of which have unusual tastes. Many of these are found in traditional food, where spontaneous plants are used as ingredients. Some taste-active compounds were identified in the bulbs of Muscari comosum, a spontaneous plant belonging to the family of the Liliaceae, very common in the Mediterranean area, and used in traditional gastronomy (called 'lampascioni' in South Italy). The bulbs were extracted with a series of solvents of different polarity. The different fractions were submitted to a preliminary sensory evaluation, and the most interesting ones, characterized by a strong bitter taste and some chemestetic properties, were submitted to further purification and structural analysis. From the ethereal extract, several 3-benzyl-4-chromanones and one stilbene derivative were isolated. Pure compounds were examined for their taste activity by means of sensory evaluation, and proved to be responsible for the characteristic taste of this food. Some of these compounds have been synthesized de novo to confirm their structure.