Use of electrochemical biosensor and gas chromatography for determination of dichlorvos in wheat

Two methods for the determination of dichlorvos in durum wheat by electrochemical assay and gas chromatography, respectively, have been developed. Dichlorvos, an organophosphorus anticholinesterase pesticide, was extracted from wheat with hexane, and the filtered extract was directly analyzed by gas chromatography with nitrogen-phosphorus flame detection (NPD). Recoveries of dichlorvos from milled wheat spiked at 0.25-1.5 microg/g ranged from 96.5 to 100.9%, and the limit of detection was 0.02 microg/g. The electrochemical assay was based on the detection of choline, the acetylcholinesterase product, via a choline oxidase biosensor. An aliquot of the filtered hexane extract was partitioned with phosphate buffer solution, and the organic layer was evaporated prior to electrochemical analysis. A limit of detection of 0.05 microg/g of dichlorvos was obtained with mean recoveries of 97-103% at spiking levels of 0.25-1.5 microg/g. A good correlation (r = 0.9919) was found between the results obtained with the electrochemical and those obtained with the gas chromatographic methods. The electrochemical method was peer-validated in two laboratories that analyzed 10 blind samples (5 duplicates), including a blank and 4 spiked samples with dichlorvos at levels of 0.25, 0.60, 1.00, and 1.50 microg/g. Within-laboratory repeatability (RSDr) and between-laboratory reproducibility (RSDR) ranged from 5.5 to 7.8% and from 9.9 to 17.6%, respectively.     

Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

Bioactive constituent production in St. John's Wort in vitro hairy roots. Regenerated plant lines

A wild strain of Agrobacterium rhizogenes was used to regenerate twelve in vitro plant lines from different hairy roots of H. perforatum (St. John's Wort). The production of the main bioactive constituents was observed even though their yields varied in the different plant lines. Two lines were selected for the hyperoside production (4.9-4.6 mg/gdw) while nine were characterized by significant yields of chlorogenic acid (ranged from 0.47 to 1.09 mg/gdw). Furthermore, one out of twelve lines showed a 10-fold higher hypericin content (0.25 mg/gdw) than that reported for the in vitro shoots in the literature. Morphological and phytochemical features were determined in order to select H. perforatum genotypes enriched in valuable bioactive compounds.     

Structure of an intermediate state in protein folding and aggregation

Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal β strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.     

Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes

The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.     

Potent antiviral flavone glycosides from Ficus benjamina leaves

Crude ethanol extracts from Ficus benjamina leaves strongly inhibit Herpes Simplex Virus 1 and 2 (HSV-1/2) as well as Varicella Zoster Virus (VZV) cell infection in vitro. Bioassay-guided fractionation of the crude extract demonstrated that the most efficient inhibition of HSV-1 and HSV-2 was obtained with the flavonoid fraction. The present study was aimed to further isolate, purify and identify substances with potent antiviral activity from the flavonoid fraction of F. benjamina extracts. Flavonoids were collected from the leaf ethanol extracts through repeated purification procedure and HPLC analysis. The antiviral activity of each substance was then evaluated in cell culture. Three known flavone glycosides, (1) quercetin 3-O-rutinoside, (2) kaempferol 3-O-rutinoside and (3) kaempferol 3-O-robinobioside, showing highest antiviral efficiency were selected and their structure was determined by spectroscopic analyses including NMR and mass spectrometry (MS). These three flavones were highly effective against HSV-1 reaching a selectivity index (SI) of 266, 100 and 666 for compound 1, 2 and 3, respectively, while the SI of their aglycons, quercetin and kaempferol amounted only in 7.1 and 3.2, respectively. Kaempferol 3-O-robinobioside showed similar SI to that of acyclovir (ACV), the standard anti-HSV drug. Although highly effective against HSV-1 and HSV-2, these flavone glycosides did not show any significant activity against VZV.     

Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.     

Snow gliding and loading under two different forest stands: a case study in the north-western Italian Alps

The presence of a thick snowpack could interfere with forest stability, especially on steep slopes with potential damages for young and old stands. The study of snow gliding in forests is rather comple...