Ku-Mediated Coupling of DNA Cleavage and Repair during Programmed Genome Rearrangements in the Ciliate Paramecium tetraurelia

During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.     

Local and Systemic Regulation of Plant Root System Architecture and Symbiotic Nodulation by a Receptor-Like Kinase

In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.     

Tree species composition and diversity in Miombo woodlands between co‐managed and government‐managed regimes,Malawi

Comparative information on the composition and diversity in tree species associations in Miombo woodland is limited. This study assessed how tree species associations across forest reserves of Miombo woodland in Malawi varied in composition and diversity concerning site factors and resource use disturbances under co‐management versus government management. Eighty nested circular plots, randomly selected in ArcGIS, were sampled to record stem diameter at breast height (DBH) of tree species: 0.04 ha for stems 5–29.9 cm DBH and 0.16 ha for stems ≥30 cm DBH. The recorded 109 tree species grouped into communities and 14 sub‐communities, using stem counts by species in TWINSPAN analysis. Sub‐divisions to level 5 showed eigenvalues ≥0.3, symbolising the stability of sub‐divisions. North/South sub‐divisions related to site factors; historical/current resource use influenced differences at levels 3–5. Species importance differed, indicating few important species in each sub‐community. Brachystegia and Julbernardia species showed importance across sub‐communities while Uapaca sansibarica in government management. Disturbances stimulated high species diversity. Recommendations include the need for a policy review towards group‐felling mature stands to stimulate regeneration and selective thinning of suppressed stems in stand development stages to maintain species diversity, productive recovery, diverse resource use value, and monitoring of harvesting impacts.     

Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44(hi)/CD24(lo) cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44(hi)/CD24(lo) cells (P < 0.05; Kruskal-Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44(hi)/CD24(lo) phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal-Wallis). Notch1 knockdown reduced the expression of CD44(hi)/CD24(lo) phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal-Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity.     

Energetic consequences of contrasting winter migratory strategies in a sympatric Arctic seabird duet

At the onset of winter, warm‐blooded animals inhabiting seasonal environments may remain resident and face poorer climatic conditions, or migrate towards more favourable habitats. While the origins and evolution of migratory choices have been extensively studied, their consequences on avian energy balance and winter survival are poorly understood, especially in species difficult to observe such as seabirds. Using miniaturized geolocators, time‐depth recorders and a mechanistic model, we investigated the migratory strategies, the activity levels and the energy expenditure of the closely‐related, sympatrically breeding Brünnich's guillemots Uria lomvia and common guillemots Uria aalge from Bjørnøya, Svalbard. The two guillemot species from this region present contrasting migratory strategies and wintering quarters: Brünnich's guillemots migrate across the North Atlantic to overwinter off southeast Greenland and Faroe Islands, while common guillemots remain resident in the Barents, the Norwegian and the White Seas. Results show that both species display a marked behavioural plasticity to respond to environmental constraint, notably modulating their foraging effort and diving behaviour. Nevertheless, we provide evidence that the migratory strategy adopted by guillemots can have important consequences for their energy balance. Overall energy expenditure estimated for the non‐breeding season is relatively similar between both species, suggesting that both southward migration and high‐arctic winter residency are energetically equivalent and suitable strategies. However, we also demonstrate that the migratory strategy adopted by Brünnich's guillemots allows them to have reduced daily energy expenditures during the challenging winter period. We therefore speculate that ‘resident’ common guillemots are more vulnerable than ‘migrating’ Brünnich's guillemots to harsh winter environmental conditions.     

The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion

During evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-β-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P. fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR.