Convergent dynamics of the juvenile European sea bass gut microbiota induced by poly-β-hydroxybutyrate

Poly-β-hydroxybutyrate (PHB) is a bacterial energy and carbon storage compound which exhibits a controlling effect on the gastrointestinal microbiota. Its beneficial activities for aquaculture have already been shown in terms of increased disease resistance and growth performance in a number of studies. However, the action of PHB on the intestinal microbial community in the treated animals has not yet been studied in depth. In this research, the effects of PHB on the microbiota composition in the intestinal tract of juvenile sea bass were examined. It was found that fish cohabiting in the same tank were on average 87% similar regarding the intestinal microbiota. When subjected to the same treatment and environmental conditions but reared in different tanks, the compositions of the enteric communities diverged. The provision of PHB overruled this tank effect by sustaining a microbial core community in the gut that represented 60% of the total bacterial diversity at the highest PHB level of 10%. The microbial community compositions converged between replicate tanks upon supplementation of PHB and were characterized by high dynamics and increased evenness. The results are discussed in the framework of hypotheses that try to relate the intestinal microbial community composition to the health status of the host organisms.     

Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor.     

Are ‘Endurance’ Alleles ‘Survival’ Alleles? Insights from the ACTN3 R577X Polymorphism

Exercise phenotypes have played a key role for ensuring survival over human evolution. We speculated that some genetic variants that influence exercise phenotypes could be associated with exceptional survival (i.e. reaching ≥100years of age). Owing to its effects on muscle structure/function, a potential candidate is the Arg(R)577Ter(X) polymorphism (rs1815739) in ACTN3, the structural gene encoding the skeletal muscle protein α-actinin-3. We compared the ACTN3 R577X genotype/allele frequencies between the following groups of ethnically-matched (Spanish) individuals: centenarians (cases, n = 64; 57 female; age range: 100–108 years), young healthy controls (n = 283, 67 females, 216 males; 21±2 years), and humans who are at the two end-points of exercise capacity phenotypes, i.e. muscle endurance (50 male professional road cyclists) and muscle power (63 male jumpers/sprinters). Although there were no differences in genotype/allele frequencies between centenarians (RR:28.8%; RX:47.5%; XX:23.7%), and controls (RR:31.8%; RX:49.8%; XX:18.4%) or endurance athletes (RR:28.0%; RX:46%; XX:26.0%), we observed a significantly higher frequency of the X allele (P = 0.019) and XX genotype (P = 0.011) in centenarians compared with power athletes (RR:47.6%; RX:36.5%;XX:15.9%). Notably, the frequency of the null XX (α-actinin-3 deficient) genotype in centenarians was the highest ever reported in non-athletic Caucasian populations. In conclusion, despite there were no significant differences with the younger, control population, overall the ACTN3 genotype of centenarians resembles that of world-class elite endurance athletes and differs from that of elite power athletes. Our preliminary data would suggest a certain ‘survival’ advantage brought about by α-actinin-3 deficiency and the ‘endurance’/oxidative muscle phenotype that is commonly associated with this condition.     

Structure of even/odd trinucleotide repeat sequences modulates persistence of non-B conformations and conversion to duplex

Expansion of trinucleotide repeats (TNR) has been implicated in the emergence of neurodegenerative diseases. Formation of non-B conformations such as hairpins by these repeat sequences during DNA replication and/or repair has been proposed as a contributing factor to expansion. In this work we employed a combination of fluorescence, chemical probing, optical melting, and gel shift assays to characterize the structure of a series of (CTG)(n) sequences and the kinetic parameters describing their interaction with a complementary sequence. Our structure-based experiments using chemical probing reveal that sequences containing an even or odd number of CTG repeats adopt stem-loop hairpins that differ from one another by the absence or presence of a stem overhang. Furthermore, we find that this structural difference dictates the rate at which the TNR hairpins convert to duplex with a complementary CAG sequence. Indeed, the rate constant describing conversion to (CAG)(10)/(CTG)(n) duplex is slower for sequences containing an even number of CTG repeats than for sequences containing an odd number of repeats. Thus, when both the CAG and CTG hairpins have an even number of the repeats, they display a longer lifetime relative to when the CTG hairpin has an odd number of repeats. The difference in lifetimes observed for these TNR hairpins has implications toward their persistence during DNA replication or repair events and could influence their predisposition toward expansion. Taken together, these results contribute to our understanding of trinucleotide repeats and the factors that regulate persistence of hairpins in these repetitive sequences and conversion to canonical duplex.     

Effect of neuropeptide S receptor antagonists and partial agonists on palatable food consumption in the rat

Neuropeptide S (NPS) is the endogenous ligand for the previously orphan G-protein-coupled-receptor, now termed NPS receptor (NPSR). NPS has both anxiolytic and pro-arousal properties and decreases food intake. In this work we use a rat model of palatable food intake to test in vivo different analogs of human NPS developed in our laboratories and characterized in previous in vitro experiments as partial agonists ([Ala³]NPS and [Aib⁵]NPS), or antagonists ([d-Cys(^tBu)⁵]NPS and [^tBu-d-Gly⁵]NPS). Our results confirmed that intracerebroventricular (ICV) injection of NPS (1 nmol) decreases standard chow intake in food restricted rats as well as in freely feeding animals fed with standard or palatable food diets. [Aib⁵]NPS (30 and 60 nmol), like NPS, reduced palatable food intake, thus confirming in vivo its ability to activate NPSR. [Ala³]NPS (60 nmol) did not affect palatable food intake per se but blocked the anorectic effect of NPS, thus suggesting its ability to function as an antagonist in this model. Finally, [d-Cys(^tBu)⁵]NPS (20-60 nmol) and [^tBu-d-Gly⁵]NPS (10-30 nmol), described in previous in vitro studies as pure NPSR antagonists, did not affect palatable food intake when given alone, but fully blocked the anorectic effect of NPS. These results provide an important characterization of the pharmacological properties of these NPS analogs in vivo. Of particular relevance are the data showing that [d-Cys(^tBu)⁵]NPS and [^tBu-d-Gly⁵]NPS behave as pure antagonists at NPSR regulating food intake, indicating that these molecules are suitable tools for further investigation of the physiopharmacology of the NPS/NPSR system.     

Guinea pig ileum motility stimulation elicited by N-formyl-Met-Leu-Phe (fMLF) involves neurotransmitters and prostanoids

In guinea-pig ileum (GPI), the chemotactic peptide N-formyl-Met-Leu-Phe-OH (fMLF) possesses spasmogenic properties through the activation of formyl peptide receptors (FPRs). Despite this, the mediators involved remain to be elucidated. fMLF (1 nM-1 μM) induced a dose-dependent contraction of GPI (EC₅₀ = 24 nM), that is blocked by pre-treatment with the FPRs antagonist Boc₂. The pre-treatment with tetrodotoxin (TTX) atropine or with SR140333 reduced the fMLF-induced contraction, whereas with hexamethonium, MEN10627, SB222200, mepyramine, cimetidine, thioperamide or methysergide did not produce any effect. With DuP697 pre-treatment, but not with piroxicam, reduced the fMLF-induced contraction. After stimulation with 24 nM fMLF, a strong increase in the PGE₂ levels was observed. Finally, the concomitant blocking of the NK₁ receptor, the muscarinic receptors and COX-2 abolished the GPI contractions induced by fMLF.fMLF induced a concentration-dependent contraction of guinea-pig jejunum (EC₅₀ = 11 nM), proximal colon (EC₅₀ = 3.5 nM) and distal colon (EC₅₀ = 2.2 nM), with a time-course similar to that observed in GPI. In these preparations as well, the co-administration of atropine, SR140333 and DuP697 abolished the contractions induced by fMLF. Intraperitoneal injection of fMLF (0.1 or 1 μmol/kg) enhanced the gastrointestinal motility in mice, abolished by the co-administration of atropine, SR140333 and DuP697. In conclusion, we showed that fMLF exerts spasmogenic actions on guinea-pig intestine both in vitro and in vivo through the release of acetylcholine and substance P from myenteric motorneurons and through prostanoids, probably from the inflammatory cells of the enteric immune system.     

PET translates neurophysiology into images: A review to stimulate a network between neuroimaging and basic research

In the last decades there has been a progressive advance in the development of techniques able to explore in humans neurophysiologic and neurochemical processes. Positron emission tomography (PET) is a very powerful technique allowing to study a quite variable range of physiological and biochemical processes in the healthy subjects and in diseases. Apart from its capacity to provide pathophysiological information, PET is also important for the objective assessment of therapeutic efficacy. Initial studies were performed measuring cerebral metabolic rate for glucose (CMRglc) and cerebral blood flow (CBF), representing an indirect index of synaptic activity. The advent of receptor tracers allowed measuring other important physiological parameters, such as receptor occupancy, and endogenous release. In neuropsychiatric disorders, as Alzheimer disease, schizophrenia, epilepsy and Huntington disease, PET has been useful to elaborate hypothesis of the pathogenesis, to relate symptoms to biological variables and to study individuals at increased risk. The new concepts of neurovascular unit and default network, preferentially active at rest, can significantly change the approach of PET, with images reflecting a complex scenario, not merely limited to neural activity, but involving the activity of the entire neurovascular unit and the multifunctional role of astrocytes. To detect dysfunction of the dialog between glutamatergic neurons and astrocytes could lead to a better understanding of altered functional brain images. In this direction a professional network between PET researchers and basic scientists, could give a determinant improvement in the capability to understand the complex physiological and pathophysiological cerebral world.     

Cl-IB-MECA enhances TRAIL-induced apoptosis via the modulation of NF-κB signalling pathway in thyroid cancer cells

Apoptosis is an endogenous process that can be a useful anti-cancer tool. This study aimed to investigate the effect of Cl-IB-MECA, adenosine receptor A3 agonist, on TRAIL-induced apoptosis of thyroid carcinoma cells. Cl-IB-MECA enhanced TRAIL-mediated apoptosis in FRO but not in ARO cells. This effect was correlated to higher expression levels of DR5 on FRO than ARO cells, that instead presented higher levels of decoy receptors, DcR1 and DcR2. To understand the cross-talk between the effect of Cl-IB-MECA and TRAIL, we evaluated the nuclear translocation of p65 and c-Rel. Since the dependency by NF-κB, TRAIL promoted the nuclear translocation of both p65 and c-Rel subunits. However, the addition of Cl-IB-MECA led to the predominant translocation of c-Rel after TRAIL addition. Furthermore, Bcl-2, cFLIP and pAkt were lower induced than caspase-3 and -9 in FRO cells. To discriminate a specific effect of TRAIL, we used tumour necrosis factor-alpha (TNF-α) with Cl-IB-MECA. In this case, no synergism was observed. In addition, the effect of Cl-IB-MECA was not A3 receptor-dependent since its antagonists, MRS1191 and FA385, failed to block Cl-IB-MECA activity on TRAIL-treated FRO cells. In conclusion, Cl-IB-MECA enhanced TRAIL-mediated apoptosis via NF-κB/c-Rel activation and DR5-dependent manner. This study may shed light on a potential drug cocktail that may prove useful as anti-cancer in an in vivo animal model. J. Cell. Physiol. 221: 378–386, 2009. © 2009 Wiley-Liss, Inc.     

Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.