Dipeptides as Leaving Group in the Enzyme‐Catalyzed DNA Synthesis

Conjugates of 2′‐deoxyadenosine monophosphate with dipeptides have been synthesized and tested as substrates for several polymerases. Although the incorporation efficiency is not very high, it demonstrates that some of these dipeptides can be accommodated in the active site of polymerases and function as leaving groups in the enzymatic synthesis of DNA.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Designing and using RNA scaffolds to assemble proteins in vivo

RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins' relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.     

ICOS ligation recruits the p50alpha PI3K regulatory subunit to the immunological synapse

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85alpha subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50alpha, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50alpha accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50alpha plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50alpha at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50alpha is known to carry a stronger lipid kinase activity compared with p85alpha. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50alpha is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

Le site de la baume Flandin (commune d’Orgnac-l’Aven) : nouvelles données sur ce gisement du Paléolithique moyen

The Baume Flandin, located in the Middle Rhone valley, yielded in the 1950s an archaeological level attributed to the Middle Palaeolithic, with abundant elongated flakes. This feature is rare in southern Europe before MIS 4. The cave was totally excavated and little evidence existed to confirm the date of this level, which was attributed to the ‘Riss–Würm’ according to the fauna remains and the sediments. New fieldwork took place in 2005, in front of the cave entrance, on the terrace. The study of the archaeological level that has been discovered brings new information on the age of the sequence of this site. The human occupation took place in the latest interglacial, at the end of isotopic stage 5e. This date leads us to revive the debate on the first evidence of the laminar debitage between northern and southern Europe.     

Ndel1 promotes axon regeneration via intermediate filaments

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.     

Fixed vs. Random Temporal Predictability of Predation Risk: An Extension of the Risk Allocation Hypothesis

Predation is a strong selective force acting on prey animals. Predation is by nature highly variable in time; however, this aspect of predation risk has traditionally been overlooked by behavioural ecologists. Lima and Bednekoff proposed the predation risk allocation hypothesis (RAH), predicting how temporal variation in predation risk drives prey antipredator behaviours. This model is based on the concept that prey adaptively allocate their foraging and antipredator efforts across high‐ and low‐risk situations, depending on the duration of high‐ vs. low‐risk situations and the relative risk associated with each of them. An unstudied extension of the RAH is the effect of predictability of predation risk. A predictable risk should lead to prey displaying minimal vigilance behaviours during predictable low‐risk periods and the strongest antipredator behaviours during risky periods. Conversely, an unpredictable predation risk should result in prey displaying constant vigilance behaviour, with suboptimal foraging rates during periods of safety but antipredator behaviours of lower intensity during periods of risk. We tested this extension of the RAH using convict cichlids exposed to high‐risk alarm cues at two frequencies of risk (1× vs. 3×) per day, on either a fixed or random schedule for 5 d. We then tested the fish for a response to high‐risk cues (alarm cues) and to low‐risk cues (disturbance resulting from the introduction of distilled water). Our study supports previous results on the effects of risk frequency and cue intensity on cichlid behaviour. We failed to show an effect of risk predictability on the behavioural responses of cichlids to high‐risk alarm cues, but predictability did influence responses to low‐risk cues. We encourage further studies to test the effect of predictability in other systems.     

Role of metalloproteinases at the onset of liver development

At the onset of liver development, the hepatic precursor cells, namely, the hepatoblasts, derive from the ventral foregut endoderm and form a bud surrounded by a basement membrane (BM). To initiate liver growth, the hepatoblasts migrate across the BM and invade the neighboring septum transversum mesenchyme. In the present study, carried out in the mouse embryo, we searched for effectors involved in this process and we examined the role of matrix metalloproteinases (MMPs). We found expression of a broad range of MMPs, among which MMP-2 was predominantly expressed in the septum transversum and MMP-14 in the hepatoblasts. Using a new liver explant culture system we showed that inhibition of MMP activity represses migration of the hepatoblasts. We conclude that MMPs are required to initiate expansion of the liver during development and that our culture system provides a new model to study hepatoblast migration.