Regenerative proliferation of differentiated cells by mTORC1‐dependent paligenosis

In 1900, Adami speculated that a sequence of context‐independent energetic and structural changes governed the reversion of differentiated cells to a proliferative, regenerative state. Accordingly, we show here that differentiated cells in diverse organs become proliferative via a shared program. Metaplasia‐inducing injury caused both gastric chief and pancreatic acinar cells to decrease mTORC1 activity and massively upregulate lysosomes/autophagosomes; then increase damage associated metaplastic genes such as Sox9; and finally reactivate mTORC1 and re‐enter the cell cycle. Blocking mTORC1 permitted autophagy and metaplastic gene induction but blocked cell cycle re‐entry at S‐phase. In kidney and liver regeneration and in human gastric metaplasia, mTORC1 also correlated with proliferation. In lysosome‐defective Gnptab^?/? mice, both metaplasia‐associated gene expression changes and mTORC1‐mediated proliferation were deficient in pancreas and stomach. Our findings indicate differentiated cells become proliferative using a sequential program with intervening checkpoints: (i) differentiated cell structure degradation; (ii) metaplasia‐ or progenitor‐associated gene induction; (iii) cell cycle re‐entry. We propose this program, which we term “paligenosis”, is a fundamental process, like apoptosis, available to differentiated cells to fuel regeneration following injury.     

ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis

The tagging‐via‐substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide‐modified farnesyl moiety and captured thanks to biotin alkyne Click‐iT® chemistry with further streptavidin‐affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C‐terminal CaaX‐box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.     

Correlation between structure of polyoxotungstates and their inhibitory activity on polymerases

The 21-tungsto-9-antimonate (TA, HPA 23), a polyoxotungstate, has shown a significant antiviral activity in vivo and in vitro. It inhibits viral and bacterial DNA polymerases. In this paper, several compounds of two polyoxotungstic families, tungstoantimonates and tungstoarsenates, have been used to specify the mechanism of polymerase inhibition. It has been demonstrated that the inhibitory activity of polyoxotungstates is not related to the occupation of their coordinative sites by cations, nor to the nature of these bound cations. Kinetic studies and binding assays have shown that polyoxotungstates bind to the polymerases in competition with the nucleic acid template. This result seems to be related to their polyanionic nature. Furthermore, the size and charge of these compounds may play a prominent part in their affinity for the polymerases.     

Clock-controlled endogenous melatonin rhythms in Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus)

The purpose of this work was to investigate the circadian melatonin system in two tropical teleost species characterized by different behavioral habits, Nile tilapia (diurnal) and African catfish (nocturnal). To do so, fish were subjected to either a control photoperiod (12L:12D), continuous light (LL) or darkness (DD), or a 6L:6D photoperiod. Under 12L:12D, plasma melatonin levels were typically low during the photophase and high during the scotophase in both species. Interestingly, in both species, melatonin levels significantly decreased prior to the onset of light, which in catfish reached similar basal levels to those during the day, demonstrating that melatonin production can anticipate photic changes probably through circadian clocks. Further evidence for the existence of such pacemaker activity was obtained when fish were exposed to DD, as a strong circadian melatonin rhythm was maintained. Such an endogenous rhythm was sustained for at least 18 days in Nile tilapia. A similar rhythm was shown in catfish, although DD was only tested for four days. Under LL, the results confirmed the inhibitory effect of light on melatonin synthesis already reported in other species. Finally, when acclimatized to a short photo-cycle (6L:6D), no endogenous melatonin rhythm was observed in tilapia under DD, with melatonin levels remaining high. This could suggest that the circadian clocks cannot entrain to such a short photocycle. Additional research is clearly needed to further characterize the circadian axis in teleost species, identify and localize the circadian clocks, and better understand the environmental entrainment of fish physiology.     

The Grey Mouse Lemur Uses Season-Dependent Fat or Protein Sparing Strategies to Face Chronic Food Restriction

During moderate calorie restriction (CR) the heterotherm Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days (winter; SD40 and SD80, respectively) and long-days (summer; LD40 and LD80, respectively) acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.     

The lectin receptor kinase LecRK-I.9 is a novel Phytophthora resistance component and a potential host target for a RXLR effector

In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a 'gain-of-susceptibility' phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar 'gain-of-susceptibility' phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process.     

Defining the risks of mesenchymal stromal cell therapy

We address the issue of the potential for malignant transformation of cultured mesenchymal stromal cells (MSC) commonly used in clinical cell-therapy protocols and describe the culture conditions under which tumorigenesis is likely to be an extremely uncommon event.     

Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells

The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin(17) only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin(17) stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin(17)-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin(17) induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein beta-catenin, partial dissociation of the complex between beta-catenin and the transmembrane protein E-cadherin, and delocalization of beta-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin(17) was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin(17). Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G(17)-Gly on the adhesion and migration of gastric epithelial cells.     

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.