Sstr2A: a relevant target for the delivery of genes into human glioblastoma cells using fiber-modified adenoviral vectors.

Glioblastomas are the most aggressive of the brain tumors occurring in adults and children. Currently available chemotherapy prolongs the median survival time of patients by only 4 months. The low efficiency of current treatments is partly owing to the blood-brain barrier, which restricts the penetration of most drugs into the central nervous system. Locoregional treatment strategies thus become mandatory. In this context, viral tools are of great interest for the selective delivery of genes into tumoral cells. Gliomas express high levels of type 2 somatostatin receptors (sstr2A), pinpointing them as suitable targets for the improvement of transduction efficiency in these tumors. We designed a new adenoviral vector based on the introduction of the full-length somatostatin (SRIF (somatotropin release-inhibiting factor)) sequence into the HI loop of the HAdV fiber protein. We demonstrate that (i) HAdV-5-SRIF uptake into cells is mediated by sstr2A, (ii) our vector drives high levels of gene expression in cells expressing endogenous sstr2A, with up to 65-fold enhancement and (iii) low doses of HAdV-5-SRIF are sufficient to infect high-grade human primary glioblastoma cells. Adenoviral vectors targeting SRIF receptors might thus represent a promising therapeutic approach to brain tumors.

Loss of medial septum cholinergic neurons in THY-Tau22 mouse model: what links with tau pathology?

Alzheimer's disease (AD) is a neurodegenerative disorder histologically defined by the cerebral accumulation of amyloid deposits and neurofibrillary tangles composed of hyperphosphorylated tau proteins. Loss of basal forebrain cholinergic neurons is another hallmark of the disease thought to contribute to the cognitive dysfunctions. To this date, the mechanisms underlying cholinergic neurons degeneration remain uncertain. The present study aimed to investigate the relationship between neurofibrillary degeneration and cholinergic defects in AD using THY-Tau22 transgenic mouse model exhibiting a major hippocampal AD-like tau pathology and hyperphosphorylated tau species in the septohippocampal pathway. Here, we report that at a time THY-Tau22 mice display strong reference memory alterations, the retrograde transport of fluorogold through the septohippocampal pathway is altered. This impairment is associated with a significant reduction in the number of choline acetyltransferase (ChAT)-immunopositive cholinergic neurons in the medial septum. Analysis of nerve growth factor (NGF) levels supports an accumulation of the mature neurotrophin in the hippocampus of THY-Tau22 mice, consistent with a decrease of its uptake or retrograde transport by cholinergic terminals. Finally, our data strongly support that tau pathology could be instrumental in the cholinergic neuronal loss observed in AD.

Milk odd- and branched-chain fatty acids in relation to the rumen fermentation pattern.

The objectives of this study were 1) to determine whether a relationship exists between molar proportions of volatile fatty acids in the rumen and milk odd-and branched-chain fatty acid concentrations (i.e., iso C13:0, anteiso C13:0, iso C14:0, C15:0, iso C15:0, anteiso C15:0, iso C16:0, C17:0, iso C17:0, anteiso C17:0, and cis-9 C17:1); and 2) to evaluate the accuracy of prediction of the latter equations using an independent data set. For development of the regression equations, individual cow data from 10 feeding experiments with rumen-fistulated dairy cows were used, resulting in a data set of 148 observations. Milk odd- and branched-chain fatty acids were closely related to the molar proportions of acetate (SE = 15.3 mmol/mol), propionate (SE = 14.7 mmol/mol), and butyrate (SE = 9.2 mmol/mol). These regression equations were further validated using data from the literature (n = 14). Evaluation of these prediction equations using the independent data set resulted in a root mean square prediction error of 3.0, 9.0, and 8.9% of the observed mean for acetate, propionate, and butyrate, respectively. In addition, less then 5% of the mean square prediction error was due to line bias. This suggests that the currently developed prediction equations based on milk odd- and branched-chain fatty acids show potential to predict molar proportions of individual volatile fatty acids in the rumen.

Effect of forage:concentrate ratio on fatty acid composition of rumen bacteria isolated from ruminal and duodenal digesta.

Four dairy cows were used to examine the effect of the dietary forage:concentrate ratio [35:65, 50:50, 65:35, and 80:20 on a dry matter (DM) basis] on the fatty acid composition of rumen bacteria isolated from the liquid (LAB) and solid (SAB) phase of the rumen and duodenal digesta. Rumen contents were sampled 4 h after the morning feeding. Solid and liquid phases were separated from rumen contents and duodenal bacteria from a composite duodenal sample by differential centrifugation. Total fatty acid content in bacterial DM was 1.6 to 2.8 times higher in SAB compared with LAB, and increased with dietary concentrate. In combination with published reports, the data show that bacterial fatty acid content and composition is closely related to dietary fatty acids except for C18:2n-6 and C18:3n-3. A decrease in forage:concentrate ratio increased bacterial concentration of trans-10 C18:1, and this increase was 3.4 times higher in LAB compared with SAB. Analysis of odd- and branched-chain fatty acids showed large differences between SAB and LAB, which probably reflected a difference in species composition. The variation in odd- and branched-chain fatty acids between SAB and LAB was used to estimate their relative proportions in duodenal bacteria by means of linear programming, and showed an increased proportion of SAB from 64.7 to 74.8% with increasing forage:concentrate ratio. In addition, increasing the proportion of dietary forage was closely related to the proportion of anteiso C15:0 in total odd- and branched-chain fatty acids (r(pearson) = -0.771). The bacterial concentration of iso C17:0 closely reflected the bacterial growth rate as shown by the relation with cytosine:N (r(pearson) = -0.729). These strong relationships suggest that odd- and branched-chain fatty acids might be used as tool to evaluate nutrient supply to rumen bacteria.

Use of odd and branched-chain fatty acids in rumen contents and milk as a potential microbial marker.

The objectives of this study were: 1) to determine if a correlation exists between rumen odd and branched-chain fatty acids (OBCFA, i.e., C(15:0), iso C(15:0), anteiso C(15:0), C(17:0), iso C(17:0), anteiso C(17:0), and C(17:1)), uracil, and purine bases (PB), 2) to evaluate the potential of milk OBCFA secretion to predict duodenal flow of microbial protein in lactating cows, 3) to evaluate the accuracy of the latter prediction equations using an independent data set, and 4) to determine whether these predictions were more accurate than predictions based on dry matter intake (DMI) and dietary characteristics. In the first experiment, 4 lactating dairy cows arranged in a 4 x 4 Latin square were offered diets based on grass silages of different botanical composition and a standard concentrate. The relationship between rumen pool size of OBCFA and microbial matter was investigated. Rumen pool size of microbial matter (g), determined 4, 12, and 17 h after feeding, using uracil and PB as microbial markers, was closely related to OBCFA (g) [r(2) = 0.716, root mean square error (RMSE) = 4.45]. To correct for differences in marker concentrations among strains of rumen microbes, C(17:0) was included in the regression equations, resulting in an increased predictive power (r(2) = 0.780, RMSE = 3.92). The relationship between microbial flow to the duodenum and milk OBCFA yield was evaluated in a second experiment with lactating dairy cows offered diets based on grass silage and concentrates differing in starch source. Similar to observations in the rumen, milk OBCFA yield was closely related to microbial flow to the duodenum (RMSE = 4.28), but predictive power of equations did not increase when straight-chain C(17)-fatty acids were included in the regression equations (RMSE = 4.92). Evaluation of the current prediction equations with 3 independent datasets resulted in a root mean square prediction error of 20.5 and 13.4% of the observed mean for equations based on milk secretion of total OBCFA and straight-chain C(17)-fatty acids, respectively. Comparison of the accuracy of the latter equations with 2 previously published equations based on DMI and dietary characteristics suggest the former to be more accurate. This first evaluation suggests that milk OBCFA could be used as a marker for duodenal flow of microbial matter, especially when accurate measurements of DMI are not available.

Rumen odd and branched chain fatty acids in relation to in vitro rumen volatile fatty acid productions and dietary characteristics of incubated substrates.

A first aim of this batch in vitro experiment (21 h) was to use changes in odd and branched chain fatty acid (OBCFA) patterns to suggest shifts in microbial populations, associated with four types of incubated whole dairy cow diets. Principal component analysis suggested higher dietary starch increased the proportion of C15:0 and C17:0, whereas increased neutral detergent fibre content was positively related to anteiso C15:0 concentrations, which is in agreement with the importance of these fatty acids in respectively amylolytic and cellulolytic bacteria. A second aim of the experiment was to relate rumen volatile fatty acid proportions to OBCFA by principal component regression and to compare these relations with predictions based on diet proximate composition. The R2 values achieved for the regressions between acetate, propionate and butyrate, and OBCFA were 79.6%, 86.6% and 84.9% respectively. Moreover, in the current study, predictions of the rumen fermentation pattern showed higher R2 (p < 0.01) when based on OBCFA compared with proximate feed composition. If relations persist in vivo, there could be scope for milk OBCFA to predict the supply of specific rumen nutrients.

Effects of freezing and drying grass products prior to fatty acid extraction on grass fatty acid and lipid class composition--a technical note.

Grass and grass silage represent a rich and natural source of omega-3 polyunsaturated fatty acids, in particular linolenic acid, for ruminants. Recent research, focusing on improving the content of these beneficial fatty acids in grass, requires storage of the forage samples prior to analysis. In this study, we evaluated whether conservation of fresh grass and grass silage by freezing (1 and 4 weeks,--18 degrees C) and/or drying (24h, 50 degrees C) affected its fatty acid content and induced shifts between lipid classes. FA were extracted using chloroform/methanol (2/1, v/v) and triacylglycerols (TAG), free fatty acids (FFA) and polar lipids (PL) were separated by thin layer chromatography. Fatty methyl esters (FAME) were identified by gas chromatography. Loss of thawing liquor might provoke a dramatic decrease in extractable lipid after frozen storage of both grass and grass silage. Morever, after frozen storage, fatty acids in grass but not in grass silage seem subjected to a higher rate o f lipolysis and oxidation, as suggested by increased quantities of FFA (3.1, 7.6, 8.4 % of total FAME) and reduced proportions of poly-unsaturated fatty acids (79.5, 73.6 and 74.1 % of total FAME) when analysing fresh grass samples directly or after 1 and 4 weeks of frozen storage, respectively. Drying of fresh grass did not provoke changes in FA composition, but distribution of FA over lipid classes was significantly altered, with an increase in TAG (5.1 to 17.9 % of total FAME) and FFA (2.4 to 14.9 % of total FAME) and lower proportions of PL (90.7 to 55.7 % of total FAME).

Nutritive value and qualitative assessment of secondary compounds in seeds of eight tropical browse, shrub and pulse legumes.

Seeds of four tropical multipurpose trees (Albizia saman, Albizia lebbeck, Albizia rhizonse, Leucaena leucocephala), two shrubs (Tephrosia candida, Tephrosia bracteolata) and two pulse legume (Lablab purpureus, Canavalia ensiformis) were chemically analysed for dry matter (DM), ash, crude protein (CP), neutral detergent fibre (NDF) and ether extract (EE). Qualitative evaluation of secondary metabolites (saponins, phenols, steroids, and alkaloids) was elucidated. The DM, ash, CP, NDF and EE ranged between 88.9-93.6 %, 3.0-5.4 %, 24.8-38.2 %, 22.1-46.9 % and 2.0-17.0 % respectively. All seed species contained at least one group of secondary plant metabolites and steroids were common to all except C. ensiformis that was not implicated for any. A. lebbeck and A. rhizonse showed low saponin content. Indications for water soluble tannins were reported for L. leucocephala while the two species of Tephrosia contained flavonoids or condensed tannins. The study suggested the potentials of the legumes seed species as a feed source for ruminants.

Quantification of fresh meat peptides by SDS-PAGE in relation to ageing time and taste intensity.

The extraction and quantification of fresh meat peptides in the molecular weight (MW) range between 3 and 17 kDalton (kDa) was examined, as well as their evolution during post-mortem ageing and their relation to taste intensity. Using a Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) method, quantification of fresh meat peptides in this MW range was possible. Correlation coefficients higher than 0.98 were found between the absolute amounts of peptides loaded on the gel and the measured amounts expressed as cytochrome c equivalents. In contrast to the extraction in the presence of SDS, extraction with 0.1 N HCl, 3% HClO(4) and 0.9% NaCl only partially recovered peptides present in fresh meat samples in both the 3-10 and 10-17 kDa MW range. A substantial increase in peptide concentration in the 3-17 kDa MW range with time post-mortem in both pork and beef fresh muscle samples was found. For grilled beef longissimus lumborum and semimembranosus samples of different origin and ripening times, both the concentration of the peptides in the 3-10 kDa MW range and the intramuscular fat content were significantly related with taste panel intensity scores, while the correlation with the amount of peptides in the 10-17 kDa MW range was not significant.

Effect of linseed feeding at similar linoleic acid levels on the fatty acid composition of double-muscled Belgian Blue young bulls.

The effect of including linseed [extruded (EL) or crushed (CL)] instead of whole soybeans (S) in the finishing diet of double-muscled Belgian Blue young bulls on the fatty acid composition of the longissimus thoracis, triceps brachii and subcutaneous fat was investigated. The dietary supply of C18:2n-6 was similar in the three diets, while in the EL and CL diet the supply of C18:3n-3 was equal. No effects of diet on the saturated, monounsaturated and branched chain fatty acids were found. Including linseed in place of whole soybeans increased the total intramuscular n-3 fatty acid content significantly, mainly as C18:3n-3, while no significant effect on the total and individual n-6 fatty acid incorporation was observed in the intramuscular fat. As a consequence of the higher n-3 content, the n-6/n-3 ratio was decreased by linseed feeding. In contrast with the intramuscular fat, the subcutaneous fat showed a significantly increased C18:3n-3 proportion accompanied by a significantly decreased C18:2n-6 proportion when linseed was fed. Diet did not influence the c9t11CLA content in the intramuscular or the subcutaneous fat.

Meat quality, fatty acid composition and flavour analysis in Belgian retail beef.

The objective of this study was to evaluate the differences in biochemical, sensorial and quality characteristics of retail beef in Belgium. Four types of beef (Belgian Blue double-muscled, Limousin, Irish and Argentine) and two different muscles (longissimus lumborum and semimembranosus) were bought at the retail level and compared with regard to colour, shear force, collagen content, fatty acid analysis, taste panel evaluation as well as flavour analysis. Belgian Blue and Limousin beef had a paler colour, lower collagen and intramuscular fat contents. Fatty acid profiles were significantly different between the four types, with significantly higher PUFA/SFA and n-6/n-3 ratios for Belgiam Blue and Limousin beef compared to Argentine and Irish beef. There were significant differences between the meat types for taste panel tenderness and shear force, however both measurements did not fully correspond. Flavour analysis by gas chromatography-mass spectrometry as well as sensory analysis demonstrated that Irish and Argentine beef had a higher flavour intensity related to higher contents of volatile compounds. Differences in tenderness and flavour between the meat types were probably affected by differences in ageing time, related to import vs local production of meat.

Analysis of biogenic amines in northern and southern European sausages and role of flora in amine production.

The biogenic amine contents, microbial counts and flora producing amines were investigated in four types of fermented sausages. Southern type European sausages (Italian and Belgian) showed higher tyramine and phenylethylamine values than northern type ones (Norwegian and Belgian). The spontaneous non-starter lactic acid bacteria could be responsible for the production of these amines in the Italian products, and the cocci Gram positive in the Belgian South ones. The Norwegian sausages showed the lowest total amine content of those studied. The two Belgian types were characterised by the highest putrescine contents, associated with high counts of Enterococcus. The production of amines in vitro by the starter cultures used in the manufacture of the sausages revealed that none of the Lactobacillus species produced any amines and only Kocuria varians and Staphylococcus carnosus showed phenylethylamine and tryptamine production. High correlations were found between the content of putrescine, histamine and cadaverine.

Effect of rate of pH decline on muscle enzyme activities in two pig lines.

Muscle enzyme activities were measured in m. transversus abdominis (TA) samples of two pig lines, differing in stress sensitivity and in breeding objectives. Animals of line one were stress sensitive and selected for a high carcass lean content (LEAN), those of line two were homozygous stress resistant and selected for maximum live weight gain (GROWTH). Muscle samples of 16 pigs per line were excised at 1 h and sampled at 3 h post mortem. Boars, gilts and barrows were almost equally represented. Pigs of line GROWTH had a higher fat and lean tissue gain (P<0.01) and a lower carcass lean content (P<0.001) than those of line LEAN. Lower activities of µ-calpain, m-calpain, pyroglutamyl aminopeptidase I, acid lipase and neutral phospholipase were measured in TA muscle samples of line LEAN compared with line GROWTH (P<0.05). Activities of dipeptidyl peptidase IV and acid phospholipase tended to be somewhat lower for line LEAN, whereas activities of calpastatin, cathepsin (B+L) and cathepsin D were not different. Sex-class effects were analogous to line effects. Significant correlation coefficients between activities of several enzymes and pH values 1 h post mortem, both within and across lines, suggest that lower enzyme activities are a consequence of a more pronounced denaturation due to a faster post mortem pH decline.