Phytochemical Profile and Nutraceutical Value of Old and Modern Common Wheat Cultivars

Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems.Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed. Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases.     

A Novel Chemotaxis Assay in 3-D Collagen Gels by Time-Lapse Microscopy

The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick''s law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.     

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP^C) into disease-specific forms, termed PrP^Sc, that have the ability to interact with PrP^C promoting its conversion to PrP^Sc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP^C region involved in the interaction with PrP^Sc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP^Sc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.     

Exploitation of the semi-homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts.     

On-column epimerization of dihydroartemisinin: an effective analytical approach to overcome the shortcomings of the International Pharmacopoeia monograph

We developed a cryo-HPLC/UV method for the simultaneous determination of artemisinin (1), alpha-dihydroartemisinin (2alpha), beta-dihydroartemisinin (2beta), and a ubiquitous thermal decomposition product of 2 (designated as diketoaldehyde, 3), starting from the International Pharmacopoeia monograph on dihydroartemisinin. The method takes for the first time the on-column epimerization process of 2 into consideration. Chromatographic separation was obtained under reversed-phase conditions on a Symmetry C18 column (3.5 microm particle size) with a mobile phase consisting of acetonitrile-water 60:40 (v/v), delivered at 0.60-1.00 ml/min flow-rates, with ultraviolet detection at low wavelength (lambda = 210 nm). Low temperatures (T = 0-10 degrees C) were selected on the grounds of a diastereoselective dynamic HPLC (DHPLC) study performed at different temperatures, aimed at identifying the best experimental conditions capable of minimizing the on-column interconversion process.     

Phytochemical analysis and in vitro antiviral activities of the essential oils of seven Lebanon species

The chemical composition of the essential oils of Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, and Satureja thymbra was determined by GC/MS analysis. Essential oils have been evaluated for their inhibitory activity against SARS-CoV and HSV-1 replication in vitro by visually scoring of the virus-induced cytopathogenic effect post-infection. L. nobilis oil exerted an interesting activity against SARS-CoV with an IC(50) value of 120 microg/ml and a selectivity index (SI) of 4.16. This oil was characterized by the presence of beta-ocimene, 1,8-cineole, alpha-pinene, and beta-pinene as the main constituents. J. oxycedrus ssp. oxycedrus oil, in which alpha-pinene and beta-myrcene were the major constituents, revealed antiviral activity against HSV-1 with an IC(50) value of 200 microg/ml and a SI of 5.     

CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.     

Phytochemical analysis and in vitro evaluation of the biological activity against herpes simplex virus type 1 (HSV-1) of Cedrus libani A. Rich.

Cedrus libani are widely used as traditional medicine in Lebanon for treatment of different infection diseases. In the present study we reported the phytochemical composition analyzed by GC-MS of wood essential oil and cones and leaves ethanol extracts. The main components of wood essential oil were himachalol (22.50%), beta-himachalene (21.90%), and alpha-himachalene (10.50%). Leaves ethanol extract was characterized by a high content of germacrene d (29.40%). The same extract obtained from cones essentially contained alpha-pinene (51.0%) and beta-myrcene (13.0%). Moreover, we investigated extracts, essential oil, and identified compounds for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1). Cytotoxicity was evaluated by MTT assay in Vero cells. Cones and leaves ethanol extracts exhibited an interesting activity with IC50 of 0.50 and 0.66 mg/ml, respectively, at non-cytotoxic concentration. A comparable activity was found when essential oil was tested (IC50 of 0.44 mg/ml).