Enhanced Peripheral Chemoreflex Drive Is Associated with Cardiorespiratory Disorders in Mice with Coronary Heart Disease.

Coronary heart disease (CHD) is a prevalent cardiovascular disease characterized by coronary artery blood flow reductions caused by lipid deposition and oxidation within the coronary arteries. Dyslipidemia is associated with local tissue damage by oxidative stress/inflammation and carotid bodies (CB) peripheral chemoreceptors are heavily modulated by both reactive oxygen species and pro-inflammatory molecules (i.e., cytokines). Despite this, it is not know whether CB-mediated chemoreflex drive may be affected in CHD. In the present study, we evaluated peripheral CB-mediated chemoreflex drive, cardiac autonomic function, and the incidence of breathing disorders in a murine model of CHD. Compared to age-matched control mice, CHD mice showed enhanced CB-chemoreflex drive (twofold increase in the hypoxic ventilatory response), cardiac sympathoexcitation, and irregular breathing disorders. Remarkably, all these were closely linked to the enhanced CB-mediated chemoreflex drive. Our results showed that mice with CHD displayed an enhanced CB chemoreflex, sympathoexcitation, and disordered breathing and suggest that CBs may be involved in chronic cardiorespiratory alterations in the setting of CHD.

Contribution of Carotid Bodies on Pulmonary Function During Normoxia and Acute Hypoxia.

Carotid bodies (CBs) are main peripheral chemoreceptors involved in breathing regulation. Despite the well-known role played by CBs on breathing control, the precise contribution of CBs on the regulation of lung mechanics remains controversial. Accordingly, we study changes in lung mechanics in normoxia (FiO2 21%) and hypoxia (FiO2 8%) in mice with or without functional CBs. For this, we used adult male mice that underwent sham or CB denervation (CBD) surgery. Compared to sham-operated mice, we found that CBD induced an increase in lung resistance (RL) while breathing normoxic air (sham vs. CBD, p < 0.05). Importantly, changes in RL were accompanied by an approximately threefold reduction in dynamic compliance (Cdyn). Additionally, end-expiratory work (EEW) was increased in normoxia in the CBD group. Contrarily, we found that CBD has no effect on lung mechanics during hypoxic stimulation. Indeed, RL, Cdyn, and EEW values in CBD mice were undistinguishable from the ones obtained in sham mice. Finally, we found that CBD induces lung parenchyma morphological alterations characterized by reduced alveoli space. Together our results showed that CBD progressively increases lung resistance at normoxic conditions and suggest that CB tonic afferent discharges are needed for the proper regulation of lung mechanics at rest.

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.

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.

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.

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.

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.