Identification and quantification of stilbenes in fruits of transgenic tomato plants (Lycopersicon esculentum Mill.) by reversed phase HPLC with photodiode array and mass spectrometry detection

Reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection was employed to study the accumulation of stilbenes and other naturally occurring polyphenol intermediates of flavonoid pathway in tomato fruits of plants genetically modified to synthesize resveratrol. The transgenic tomato fruits were obtained by overexpression of a grapevine gene encoding the enzyme stilbene synthase in tomato plants (Lycopersicon esculentum Mill.). Stilbenes and flavonoids, either glycosylated or free, were simultaneosly identified by electrospray interface (ESI)-MS in negative ionization mode and were quantified by PDA detection at the wavelength corresponding to their maximum absorbance. The two detectors were coupled online with an HPLC system utilizing a narrow-bore C18 reversed-phase column, which was eluted by a multistep gradient of increasing concentration of acetonitrile in water containing 0.5% (v/v) formic acid. The results of these analysis revealed that the genetic modification of the tomato plants originated different levels of accumulation of four stilbenes (i.e., trans- and cis-piceid and trans- and cis-resveratrol) in their fruit depending on the stages of ripening. Either at immature or at mature stages of ripening the stilbenes were preferentially accumulated in the fruit peel as the glycosylated form. The highest amount of trans-piceid and trans-resveratrol were found in the peel of fruits harvested at mature stage of ripening. The variations in the levels of rutin, naringenin, and chlorogenic acid found in the samples extracted from the fruits of transgenic tomato plants, in comparison to that determined in the control lines, seemed to be related to the genetic transformation, whose effect on the flavonoid biosynthetic pathway needs to be elucidated by additional studies.     

In vitro and in silico analysis of two genes (A2-1 and G1-1) differentially regulated during dormancy and sprouting in potato tubers

Summary Two cDNAs, corresponding to genes differentially regulated during dormancy and sprouting in potato tubers, cultivar Désirée, were isolated: i) G1-1 corresponded to a gene that was turned off during dormancy and turned on during early phases of sprouting; ii) A2-1 corresponded to a gene activated during dormancy and strongly repressed during the transition from dormancy to sprouting. When induced, both genes were expressed at low level. Full-length cDNAs and genomic clones were isolated and characterized. G1-1 was a short gene, 452 bp long, containing an intronless open reading frame, coding for a putative protein of 64 aminoacids. Sequence analysis showed that G1-1 was homologous to an expressed sequence tag (EST) ofArabidopsis thaliana. A2-1 full-length cDNA was 1577 bp long and contained an open reading frame coding for a putative protein of 383 aminoacids, which contained a Walker box binding domain, common to a multifunctional family of intracellular ATPases.     

Silencing of G1-1 and A2-1 genes. Effects on general plant phenotype and on tuber dormancy inSolanum tuberosum L.

Summary We have evaluated the effect that the, silencing of two genes specifically expressed in conditions of dormancy (A2-1) and sprouting (G1-1) had on tuber dormancy. For this purpose potato plants (Solanum tuberosum L. cv. Désirée) were transformed with the antisense of the genes G1-1 and A2-1 under the control of constitutive 35S CaMV promoter. A first generation of transgenic plants was propagated from axenic stem cuttings and a second generation by tuber planting. The plants obtined were analyzed for the length of dormancy, plant morphology and agronomic characteristics. Statistical analysis of dormancy in lines obtained from the original transformants for the antisense of G1-1 gene showed a significant increase in length as compared with different types of control plants, with few effects on plant vegetative habit and tuber production. In contrast, results obtained on A2-1 antisense transformed plants did not reveal any significant change on the length of dormancy. Here we report small-scale field trials performed with the aim to select and regenerate commercially exploitable potato plants with a stable transgene-dependent phenotype, affecting the length of dormancy.     

Biomaterials and Bioactive Natural Products from Marine Invertebrates: From Basic Research to Innovative Applications

Aquatic invertebrates are a major source of biomaterials and bioactive natural products that can find applications as pharmaceutics, nutraceutics, cosmetics, antibiotics, antifouling products and biomaterials. Symbiotic microorganisms are often the real producers of many secondary metabolites initially isolated from marine invertebrates; however, a certain number of them are actually synthesized by the macro-organisms. In this review, we analysed the literature of the years 2010–2019 on natural products (bioactive molecules and biomaterials) from the main phyla of marine invertebrates explored so far, including sponges, cnidarians, molluscs, echinoderms and ascidians, and present relevant examples of natural products of interest to public and private stakeholders. We also describe omics tools that have been more relevant in identifying and understanding mechanisms and processes underlying the biosynthesis of secondary metabolites in marine invertebrates. Since there is increasing attention on finding new solutions for a sustainable large-scale supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of aquatic invertebrate stem cells.     

Pulsed electric field-assisted vinification of aglianico and piedirosso grapes

Pulsed electric field (PEF) treatments were applied to increase the polyphenolic content of fresh red wines made from Aglianico and Piedirosso grapes. Prior to the fermentation/maceration step, the grape skins were treated at different PEF intensities (field strengths from 0.5 to 1.5 kV/cm and energy inputs from 1 to 50 kJ/kg), with their permeabilization being characterized by electrical impedance measurements. Furthermore, the release kinetics of the total polyphenols and anthocyanins were characterized during the maceration stage by spectroscopic and Folin-Ciocalteu colorimetric methods, respectively. Finally, the fresh wine, obtained after pressing, was characterized for total acidity, pH, reducing sugar, color intensity, total polyphenols, anthocyanins content, antioxidant activity, and volatile compound composition. PEF treatment on Aglianico grapes induced a significantly higher release of polyphenols (+20%) and anthocyanins (+75%), thus improving the color intensity (+20%) and the antioxidant activity of the wine (+20%) while preserving the other organoleptic characteristics. In contrast, there was only a minor impact on the polyphenolic release kinetics of Piedirosso grapes, despite the significant degree of cell membrane permeabilization.     

Protein haze formation in white wines: effect of saccharomyces cerevisiae cell wall components prepared with different procedures

Cell wall material was extracted by five different methods from an oenological strain of Saccharomyces cerevisiae. Enzyme preparations containing beta-glucanase activity (Zymolyase, Glucanex, and Finizym 250L) allowed a better extraction yield compared to that of dithiothreitol (DTT) and EDTA. The yeast extracts were only soluble in part in wine. The wine-soluble fraction (WSF) of the five extracts, differing in both protein and sugar contents, when added in increasing amounts to white wine differently affected protein haze formation, as determined by the heat test, giving dose/response curves of different shapes. The curves obtained with the WSF derived from DDT and Zymolyase extracts showed a plateau value corresponding to 90% and 80% of wine haze reduction, respectively. In contrast, addition of the WSF derived from the other extracts resulted in increased turbidity with respect to the original wine. The mannoproteins (MP), isolated from each yeast extract by Concanavalin-A chromatography, gave dose/response curves showing shapes more similar among them than those obtained with the whole WSFs. The best wine stabilization was obtained with the MP of the DTT and Zymolyase extracts. The supernatants obtained after heating the MP of the different extracts were also tested. The stabilizing effect of the heat-stable MP (HSMP) was always larger than that of the corresponding total (un-heated) MP. The HSMP obtained starting from the DTT and Zymolyase extracts showed the best haze-protecting effect, which was, however, lower than that obtained with their corresponding WSF. This result suggests that wine protein stabilization by compounds of the yeast cell wall could be related, in addition to the action of the MP, also to the presence of other substances of different nature.     

Genetic diversity and pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolated from wilted rocket plants in Italy

Thirty-six isolates of Fusarium oxysporum originated from Eruca vesicaria and Diplotaxis tenuifolia together with eight reference strains belonging to the formae speciales raphani, matthioli and conglutinans, typical on the Brassicaceae family, were tested for pathogenicity on two species of rocket plants (E. vesicaria L., syn. E. sativa, cv. ‘Rucola coltivata’; and D. tenuifolia cv. ‘Winter’) cultivated in the glasshouse. The results showed that different isolates were slightly, moderately or highly virulent. The strains were examined for differences in the nucleotide sequence of the ribosomal DNA (rDNA) intergenic spacer (IGS) region, about 2.5 kb long. The phylogenetic (neighbor-joining) analysis performed on the isolates enabled identification of four different groups, named I, II, III and IV. Thirty-one isolates out of 36 clustered in group I and were genetically similar to F. oxysporum f.sp. raphani. By considering the pathogenicity of the strains included in Group I, a partial host specialization could be observed: the average disease index of the isolates from D. tenuifolia was higher on wild rocket, whereas the average disease index of the isolates from E. vesicaria was higher on cultivated rocket. Moreover, isolates from cultivated rocket showed, on average, a higher degree of aggressiveness than the isolates from wild rocket. Concerning Group I, the sequence analysis confirmed the homogeneity of the population, with only five parsimony-informative SNPs and five haplotypes. Twenty-six out of 31 isolates belonged to haplotype 1. Groups II and III were genetically similar to strains of F. oxysporum f.sp. matthioli. Three other strains, not pathogenic or with a medium level of virulence, clustered together in Group 4, but their sequence was distant from that of other formae speciales. The pathogenicity and IGS analysis confirmed the presence of virulence variation and genetic diversity among the F. oxysporum isolates studied. To our knowledge, this is the first report of differentiation of formae speciales of F. oxysporum on rocket plants by IGS analysis.     

Reproduction in porcine circovirus type 2 (PCV2) seropositive gilts inseminated with PCV2b spiked semen

Background

Since 1999, field evidence of transplacental infection by porcine circovirus type 2 (PCV2) and reproductive failure has been reported in pigs. The objective of this study was to evaluate the clinical and pathological consequences of PCV2 infection in conventional PCV2-seropositive gilts by insemination with PCV2b-spiked semen.

Results

Six PCV2 seropositive gilts were inseminated with PCV2b-supplemented semen (infected) and three animals with semen and cell culture medium (controls). Only three out of the six infected animals were pregnant by ultrasonography on day 29 after insemination, while two out of the three controls were pregnant. One control gilt aborted on day 23 after insemination but not due to PVC2. Viraemia was demonstrated in four out of six infected and in one control gilt that became infected with PCV2a. Anti-PCV2 antibody titres showed dynamic variations in the infected group throughout the study. Among infected gilts, the animal with the lowest anti-PCV2 titre (1/100) at the beginning of the experiment and another that reached a similar low value during the experiment showed evident seroconversion over time and had also PCV2 positive foetuses. One placenta displayed mild focal necrosis of the chorionic epithelium positively stained by immunohistochemistry for PCV2 antigen.

Conclusions

PCV2-seropositive gilts can be infected with PCV2 after intrauterine exposure and low maternal antibody titre may increase the probability of a foetal infection.     

Administering an appeasing substance to beef calves at weaning to optimize productive and health responses during a 42-d preconditioning program

This experiment evaluated the impacts of administering a bovine appeasing substance (BAS) to beef calves at weaning on their performance, physiological responses, and behavior during a 42-d preconditioning program. Eighty calves (40 heifers and 40 steers; 90% British × 10% Nellore) were weaned at 233 ± 2 d of age (day 0); ranked by sex, weaning age, and body weight (BW); and assigned to receive BAS (IRSEA Group, Quartier Salignan, France; n = 40) or placebo (diethylene glycol monoethyl ether; CON; n = 40). Treatments (5 mL) were topically applied to the nuchal skin area of each animal following dam separation. Within treatment, calves were allocated to one of eight drylot pens (four pens per treatment; pen being the experimental unit) and received a free-choice total mixed ration (TMR) from day 0 to 42, intake of which was assessed daily. Live behavior observations were conducted on days 1, 2, 4, 8, 16, and 32. Temperament was assessed and blood samples were collected via jugular venipuncture on days −21, 0, 3, 7, 14, 28, and 42. Hair samples were collected from the tail switch on days 0, 14, 28, and 42. Calves were vaccinated against bovine respiratory disease viruses on days −21 and 0. Average daily gain from day 0 to 42 did not differ between treatments (P = 0.57) but was greater (P = 0.05) in BAS vs. CON calves from day 0 to 28. Intake of TMR was greater (P = 0.05) during the first week for BAS vs. CON calves (treatment × week; P = 0.08). The mean proportion of calves feeding simultaneously and performance of social and play behaviors were greater (P ≤ 0.05) for BAS vs. CON calves. Escape attempts were greater (P < 0.01) for BAS vs. CON calves on day 1 (treatment × day; P = 0.03). Exit velocity was greater (P = 0.04) for CON vs. BAS calves on day 14 and tended (P = 0.10) to be greater for CON vs. BAS calves on day 7 (treatment × day; P = 0.03). Mean plasma concentrations of haptoglobin were greater (P = 0.02) in CON vs. BAS calves. Hair cortisol concentrations were greater (P = 0.05) in CON vs. BAS calves on day 14 (treatment × day; P = 0.03). Mean serum concentrations of antibodies against bovine viral diarrhea virus were greater (P = 0.02) in BAS vs. CON calves. Collectively, BAS administration to beef calves at weaning alleviated stress-induced physiological reactions, improved temperament evaluated via chute exit velocity, enhanced humoral immunity acquired from vaccination, and appeared to have accelerated adaptation to novel management scheme and environment.     

Effects of clinical mastitis and puerperal diseases on reproductive efficiency of dairy cows

Tropical Animal Health and Production - The objective of this study was to evaluate the effects of clinical mastitis (CM) occurring before or after the first AI postpartum, and puerperal diseases...