Lipid-Encapsuled Grape Tannins Prevent Oxidative-Stress-Induced Neuronal Cell Death, Intracellular ROS Accumulation and Inflammation.

The central nervous system (CNS) is particularly vulnerable to oxidative stress and inflammation, which affect neuronal function and survival. Nowadays, there is great interest in the development of antioxidant and anti-inflammatory compounds extracted from natural products, as potential strategies to reduce the oxidative/inflammatory environment within the CNS and then preserve neuronal integrity and brain function. However, an important limitation of natural antioxidant formulations (mainly polyphenols) is their reduced in vivo bioavailability. The biological compatible delivery system containing polyphenols may serve as a novel compound for these antioxidant formulations. Accordingly, in the present study, we used liposomes as carriers for grape tannins, and we tested their ability to prevent neuronal oxidative stress and inflammation. Cultured catecholaminergic neurons (CAD) were used to establish the potential of lipid-encapsulated grape tannins (TLS) to prevent neuronal oxidative stress and inflammation following an oxidative insult. TLS rescued cell survival after H2O2 treatment (59.4 ± 8.8% vs. 90.4 ± 5.6% H2O2 vs. TLS+ H2O2; p < 0.05) and reduced intracellular ROS levels by ~38% (p < 0.05), despite displaying negligible antioxidant activity in solution. Additionally, TLS treatment dramatically reduced proinflammatory cytokines' mRNA expression after H2O2 treatment (TNF-α: 400.3 ± 1.7 vs. 7.9 ± 1.9-fold; IL-1ß: 423.4 ± 1.3 vs. 12.7 ± 2.6-fold; p < 0.05; H2O2 vs. TLS+ H2O2, respectively), without affecting pro/antioxidant biomarker expression, suggesting that liposomes efficiently delivered tannins inside neurons and promoted cell survival. In conclusion, we propose that lipid-encapsulated grape tannins could be an efficient tool to promote antioxidant/inflammatory cell defense.

Progress and challenges in developing organoids in farm animal species for the study of reproduction and their applications to reproductive biotechnologies.

Within the past decades, major progress has been accomplished in isolating germ/stem/pluripotent cells, in refining culture medium and conditions and in establishing 3-dimensional culture systems, towards developing organoids for organs involved in reproduction in mice and to some extent in humans. Haploid male germ cells were generated in vitro from primordial germ cells. So were oocytes, with additional support from ovarian cells and subsequent follicle culture. Going on with the female reproductive tract, spherical oviduct organoids were obtained from adult stem/progenitor cells. Multicellular endometrial structures mimicking functional uterine glands were derived from endometrial cells. Trophoblastic stem cells were induced to form 3-dimensional syncytial-like structures and exhibited invasive properties, a crucial point for placentation. Finally, considering the embryo itself, pluripotent embryonic cells together with additional extra-embryonic cells, could self-organize into a blastoid, and eventually into a post-implantation-like embryo. Most of these accomplishments have yet to be reached in farm animals, but much effort is devoted towards this goal. Here, we review the progress and discuss the specific challenges of developing organoids for the study of reproductive biology in these species. We consider the use of such organoids in basic research to delineate the physiological mechanisms involved at each step of the reproductive process, or to understand how they are altered by environmental factors relevant to animal breeding. We evaluate their potential in reproduction of animals with a high genetic value, from a breeding point of view or in the context of preserving local breeds with limited headcounts.

Spray drying encapsulation of a native plant extract rich in phenolic compounds with combinations of maltodextrin and non-conventional wall materials.

Crotalaria longirostrata (chipilin) leaves contain phenolic compounds with antioxidant activity. These phenolic compounds, however, could easily degrade after extraction. Microencapsulation is a possible solution for avoiding this degradation. Frequently, microencapsulation is carried out using conventional encapsulating agents. The aim of this work was to evaluate the effect of several non-conventional encapsulating agents on microencapsulation by spray drying of phenolic compounds from chipilin, stability and release of phenolic compounds were also studied. Maltodextrin (MD), gum Arabic (GA), soy protein (SP), cocoa shell pectin (CSP), and protein (PC), as well as the gum (GC) of Cajanus cajan seeds were used. Different blends of these matrixes containing phenolic compounds from chipilin leaves were spray dried at 120 °C. After drying, the yield and microencapsulation efficiency were determined. All results were analyzed by an ANOVA test (p < 0.05). The release kinetics of phenolic compounds were modeled using zero, first-order, Higuchi and Korsmeyer-Peppas models. The R2 was calculated for each model. The blends of encapsulating agents allowed the formation of an efficient polymer matrix with yields between 46 and 64% and microencapsulation efficiency between 65 and 92%. Results show that maltodextrin with soy protein allowed the highest (92%) microencapsulation efficiency, although maltodextrin and cocoa shell pectin were more effective protective agents, showing greater stability. The Korsmeyer-Peppas model was the best in predicting the phenolic compounds release with R2 values higher than 98%. The stability time for microcapsules with MD-CSP was 8.88 years and 1.43 years at 4 °C and 30 °C, respectively.

Stress associated with hospitalization in patients with COPD: the role of social support and health related quality of life.

BACKGROUND: The objective of this study was to determine stress levels during hospitalization in patients with Chronic Obstructive Pulmonary Disease (COPD). We wanted to relate stress to previous level of quality of life and patients' Social Support. METHODS: 80 patients (70.43; SD = 8.13 years old) with COPD were assessed by means of: Hospital Stress Rating Scale, Nottingham Health Profile, St. George's Respiratory Questionnaire and Social Support Scale. RESULTS: COPD patients' stress levels are lower than expected independently from the severity or number of previous hospitalizations. Linear regression analysis shows the predictive value of Quality of Life and Social Support on stress level during hospitalization (p < 0.0001). CONCLUSION: HRQOL and social support can be associated with stress during hospitalization.

The promoter region of the Zinnia elegans basic peroxidase isoenzyme gene contains cis-elements responsive to nitric oxide and hydrogen peroxide.

NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.

Variations of N-acetylation level of peptidoglycan do not influence persistence of Lactococcus lactis in the gastrointestinal tract.

The food-grade Gram-positive bacterium, Lactococcus lactis, is recognized as a potential candidate to deliver proteins of medical interest by mucosal routes. The ability of carrier bacteria to persist and/or to lyse in the gastrointestinal tract needs to be considered to design optimal carrier strains to deliver proteins of interest at the mucosal level. Meyrand et al. (2007) have previously characterized in L. lactis, a peptidoglycan (PG) N-acetylglucosamine deacetylase (PgdA), which activity on PG influences bacterial sensitivity to lysozyme. Inactivation of pgdA gene in this bacterium, led to fully acetylated PG, resulting in a lysozyme-sensitive phenotype, whereas pgdA overexpression led to an increased degree of PG deacetylation, resulting in a lysozyme-resistant phenotype (Meyrand et al., 2007). In order to determine whether variations in L. lactis resistance to host lysozyme may influence its persistence in the GIT and its ability to deliver heterologous proteins in situ, we constructed L. lactis strains with different de-N-acetylation levels and producing a model antigen (the human papillomavirus type-16 E7 protein) and we compared the pharmacokinetics properties of these recombinant strains with that of a wild-type strain producing the same antigen in the GIT of mice. Our results show that there was no correlation between survival, at the ileum level, of bacteria intragastrically administered in mice and bacteria sensitivity or resistance to lysozyme. In addition, analysis of the E7-specific immune response evoked by the three strains after mucosal administration in mice suggest that neither lysozyme-sensitive nor lysozyme-resistant phenotype in L. lactis enhances significantly the potential of this bacterium as mucosal delivery live vector. In conclusion, our results suggest that either pgdA inactivation or pgdA overexpression in L. lactis leading to different levels of PG deacetylation does not confer any advantage in the persistence of this bacterium in the GIT and its ability to enhance host immune responses induced by delivered antigen in situ.

A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice.

The present study demonstrates for the first time the transfer of vancomycin resistance (vanA cluster) from enterococci to a Lactobacillusacidophilus commercial strain. Transfers were observed in vitro, but also in vivo in the gut of mice (in the absence of antibiotic pressure) where transconjugants arose at relatively high frequencies and could persist in the digestive environment. Since transfer of vancomycin resistance genes might also take place in the human digestive tract, lactobacilli probiotics should be carefully considered especially in either immunocompromised patients or during antibiotherapy. Acquisition and retransfer of resistance genes should be addressed in the safety evaluation of probiotics.

Transfer of plasmid and chromosomal glycopeptide resistance determinants occurs more readily in the digestive tract of mice than in vitro and exconjugants can persist stably in vivo in the absence of glycopeptide selection.

OBJECTIVES AND METHODS: The transferability of vanA and vanB glycopeptide resistance determinants with a defined plasmid (n = 9) or chromosomal (n = 4) location between Enterococcus faecium strains of human and animal origins was compared using filter mating (in vitro) and germ-free mice (in vivo) as experimental models. Moreover, the stability of exconjugants in vivo in the absence of antibiotic selection was examined. RESULTS: Higher transfer rates were observed in vivo for four of six vanA and five of six vanB donor strains. For plasmid-encoded resistance, several log higher transfer frequencies were observed in vivo for some strains. Moreover, the in vivo model supported transfer of plasmid-encoded vanB (1 x 10(-7) exconjugants/donor) when repeated in vitro experiments were negative (estimated < 1 x 10(-9) exconjugants/donor). Readily detectable transfer of plasmid-located vanA and vanB as well as large chromosomal (>200 kb) vanB elements was observed after 24 h. The number of plasmid-mediated vanA exconjugants generally decreased markedly after 3 days. However, exconjugants containing a plasmid harbouring the vanA transposon Tn1546 linked to the post-segregational killing system omega-epsilon-zeta persisted stably in vivo in the absence of glycopeptides for more than 20 days. CONCLUSIONS: The overall results support the notion that the in vitro model underestimates the transfer potential. Rapid transfer of vanA plasmids from poultry- and pig-derived strains to human faecal E. faecium shows that even transiently colonizing strains may provide a significant reservoir for transfer of resistance genes to the permanent commensal flora. Newly acquired resistance genes may be stabilized and persist in new populations in the absence of antibiotic selection.

RuvAB is essential for replication forks reversal in certain replication mutants.

Inactivated replication forks may be reversed by the annealing of leading- and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.

Evidence of vancomycin resistance gene transfer between enterococci of human origin in the gut of mice harbouring human microbiota.

OBJECTIVES: Potential intra- and inter-species transfers of vancomycin resistance genes (vanA gene cluster) between Enterococcus strains were evaluated in the gut of heteroxenic mice harbouring a human microbiota. METHODS: Mice colonized with a stable population of E. faecium 64/3 or E. faecalis JH2-2 recipient strain and harbouring an enterococci-free human microbiota were obtained. Donor strain E. faecium HC-VI2 of clinical origin was administered orogastrically to these mice and transfers were evaluated over time in faecal samples. RESULTS: Only intraspecies transfers were detected in the digestive tract (DT) of mice harbouring a human microbiota. E. faecium 64/3 transconjugants were detected at several sampling times over the 60 day experiment to levels up to 10(3) cfu/g of faeces, but they did not steadily colonize the DT. CONCLUSIONS: Here, we show for the first time that transfer of the vanA gene cluster can occur between Enterococcus strains in the DT colonized with a human microbiota and in the absence of selective pressure. The colonization properties of other enterococci transconjugants and the influence of vancomycin intake should be further investigated since transfers in the DT of animals and humans might contribute to emergence and dissemination of new vancomycin-resistant bacteria.

A fork-clearing role for UvrD.

The inactivation of a replication protein causes the disassembly of the replication machinery and creates a need for replication reactivation. In several replication mutants, restart occurs after the fork has been isomerized into a four-armed junction, a reaction called replication fork reversal. The repair helicase UvrD is essential for replication fork reversal upon inactivation of the polymerase (DnaE) or the beta-clamp (DnaN) subunits of the Escherichia coli polymerase III, and for the viability of dnaEts and dnaNts mutants at semi-permissive temperature. We show here that the inactivation of recA, recFOR, recJ or recQ recombination genes suppresses the requirement for UvrD for replication fork reversal and suppresses the lethality conferred by uvrD inactivation to Pol IIIts mutants at semi-permissive temperature. We propose that RecA binds inappropriately to blocked replication forks in the dnaEts and dnaNts mutants in a RecQ- RecJ- RecFOR-dependent way and that UvrD acts by removing RecA or a RecA-made structure, allowing replication fork reversal. This work thus reveals the existence of a futile reaction of RecA binding to blocked replication forks, that requires the action of UvrD for fork-clearing and proper replication restart.

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal transit of healthy volunteers consuming yogurt.

To date, there is significant controversy as to the survival of yogurt bacteria (namely, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) after passage through the human gastrointestinal tract. Survival of both bacterial species in human feces was investigated by culture on selective media. Out of 39 samples recovered from 13 healthy subjects over a 12-day period of fresh yogurt intake, 32 and 37 samples contained viable S. thermophilus (median value of 6.3 x 10(4) CFU g(-1) of feces) and L. delbrueckii (median value of 7.2 x 10(4)CFU g(-1) of feces), respectively. The results of the present study indicate that substantial numbers of yogurt bacteria can survive human gastrointestinal transit.

The DNA repair helicase UvrD is essential for replication fork reversal in replication mutants.

Replication forks arrested by inactivation of the main Escherichia coli DNA polymerase (polymerase III) are reversed by the annealing of newly synthesized leading- and lagging-strand ends. Reversed forks are reset by the action of RecBC on the DNA double-strand end, and in the absence of RecBC chromosomes are linearized by the Holliday junction resolvase RuvABC. We report here that the UvrD helicase is essential for RuvABC-dependent chromosome linearization in E. coli polymerase III mutants, whereas its partners in DNA repair (UvrA/B and MutL/S) are not. We conclude that UvrD participates in replication fork reversal in E. coli.

Multiple pathways process stalled replication forks.

Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability. Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery. Several strategies for the processing of inactivated replication forks before replisome reassembly have been described. Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest. The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage. These various restart pathways may reflect different structures at stalled forks. We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes.

Primosome assembly requirement for replication restart in the Escherichia coli holDG10 replication mutant.

In this report, we study the role of pre-primosome proteins in a strain in which the frequency of replication arrest is increased because of a mutation in a replication protein. The holDG10 mutant was used, in which replication restart involves replication fork reversal. As expected, PriA primosome assembly function is essential for growth of the holDG10 mutant. The priA300 mutation, which inactivates only the helicase function of PriA in vitro, and priB inactivation strongly impair viability. In contrast, priC inactivation has no effect. Therefore, PriB is more important than PriC for PriA-dependent replication fork restart in vivo. The gain of function mutation dnaC809 restores the viability of holDG10 priA and holDG10 priB mutants only to some extent. The dnaC809 820 double mutation restores full viability to the holDG10 mutant lacking either PriA or PriB. Similarly to the holDG10 single mutant, the holDG10 priA dnaC809 820 strain is depend-ent on RecBC for viability, indicating that facilitating primosome assembly using the dnaC809 820 mutation does not allow bypass of replication fork reversal.