Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation,and impacts on metastasis free survival in breast cancer patients

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.     

Early Standard Electroencephalogram Abnormalities Predict Mortality in Septic Intensive Care Unit Patients

IntroductionSepsis is associated with increased mortality, delirium and long-term cognitive impairment in intensive care unit (ICU) patients. Electroencephalogram (EEG) abnormalities occurring at the acute stage of sepsis may correlate with severity of brain dysfunction. Predictive value of early standard EEG abnormalities for mortality in ICU septic patients remains to be assessed.MethodsIn this prospective, single center, observational study, standard EEG was performed, analyzed and classified according to both Synek and Young EEG scales, in consecutive patients acutely admitted in ICU for sepsis. Delirium, coma and the level of sedation were assessed at the time of EEG recording; and duration of sedation, occurrence of in-ICU delirium or death were assessed during follow-up. Adjusted analyses were carried out using multiple logistic regression.ResultsOne hundred ten patients were included, mean age 63.8 (±18.1) years, median SAPS-II score 38 (29–55). At the time of EEG recording, 46 patients (42%) were sedated and 22 (20%) suffered from delirium. Overall, 54 patients (49%) developed delirium, of which 32 (29%) in the days after EEG recording. 23 (21%) patients died in the ICU. Absence of EEG reactivity was observed in 27 patients (25%), periodic discharges (PDs) in 21 (19%) and electrographic seizures (ESZ) in 17 (15%). ICU mortality was independently associated with a delta-predominant background (OR: 3.36; 95% CI [1.08 to 10.4]), absence of EEG reactivity (OR: 4.44; 95% CI [1.37–14.3], PDs (OR: 3.24; 95% CI [1.03 to 10.2]), Synek grade ≥ 3 (OR: 5.35; 95% CI [1.66–17.2]) and Young grade > 1 (OR: 3.44; 95% CI [1.09–10.8]) after adjustment to Simplified Acute Physiology Score (SAPS-II) at admission and level of sedation. Delirium at the time of EEG was associated with ESZ in non-sedated patients (32% vs 10%, p = 0.037); with Synek grade ≥ 3 (36% vs 7%, p< 0.05) and Young grade > 1 (36% vs 17%, p< 0.001). Occurrence of delirium in the days after EEG was associated with a delta-predominant background (48% vs 15%, p = 0.001); absence of reactivity (39% vs 10%, p = 0.003), Synek grade ≥ 3 (42% vs 17%, p = 0.001) and Young grade >1 (58% vs 17%, p = 0.0001).ConclusionsIn this prospective cohort of 110 septic ICU patients, early standard EEG was significantly disturbed. Absence of EEG reactivity, a delta-predominant background, PDs, Synek grade ≥ 3 and Young grade > 1 at day 1 to 3 following admission were independent predictors of ICU mortality and were associated with occurence of delirium. ESZ and PDs, found in about 20% of our patients. Their prevalence could have been higher, with a still higher predictive value, if they had been diagnosed more thoroughly using continuous EEG.     

Tree Species Composition and Harvest Intensity Affect Herbivore Density and Leaf Damage on Beech,Fagus sylvatica,in Different Landscape Contexts

Most forests are exposed to anthropogenic management activities that affect tree species composition and natural ecosystem processes. Changes in ecosystem processes such as herbivory depend on management intensity, and on regional environmental conditions and species pools. Whereas influences of specific forest management measures have already been addressed for different herbivore taxa on a local scale, studies considering effects of different aspects of forest management across different regions are rare. We assessed the influence of tree species composition and intensity of harvesting activities on arthropod herbivores and herbivore-related damage to beech trees, Fagus sylvatica, in 48 forest plots in three regions of Germany. We found that herbivore abundance and damage to beech trees differed between regions and that – despite the regional differences - density of tree-associated arthropod taxa and herbivore damage were consistently affected by tree species composition and harvest intensity. Specifically, overall herbivore damage to beech trees increased with increasing dominance of beech trees – suggesting the action of associational resistance processes – and decreased with harvest intensity. The density of leaf chewers and mines was positively related to leaf damage, and several arthropod groups responded to beech dominance and harvest intensity. The distribution of damage patterns was consistent with a vertical shift of herbivores to higher crown layers during the season and with higher beech dominance. By linking quantitative data on arthropod herbivore abundance and herbivory with tree species composition and harvesting activity in a wide variety of beech forests, our study helps to better understand the influence of forest management on interactions between a naturally dominant deciduous forest tree and arthropod herbivores.     

Functional diversification in the Nudix hydrolase gene family drives sesquiterpene biosynthesis in Rosa × wichurana

Roses use a non‐canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1‐2. In order to study the function of the RwNUDX1‐2 protein, we analyzed the volatile profiles of an F₁ progeny generated by crossing R. chinensis cv. ‘Old Blush’ with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1‐2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E‐farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1‐2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E‐farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1‐2 gene co‐localized with the QTL for E,E‐farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.     

Contrasting genomic and phenotypic outcomes of hybridization between pairs of mimetic butterfly taxa across a suture zone

Hybrid zones, whereby divergent lineages come into contact and eventually hybridize, can provide insights on the mechanisms involved in population differentiation and reproductive isolation, and ultimately speciation. Suture zones offer the opportunity to compare these processes across multiple species. In this paper we use reduced‐complexity genomic data to compare the genetic and phenotypic structure and hybridization patterns of two mimetic butterfly species, Ithomia salapia and Oleria onega (Nymphalidae: Ithomiini), each consisting of a pair of lineages differentiated for their wing colour pattern and that come into contact in the Andean foothills of Peru. Despite similarities in their life history, we highlight major differences, both at the genomic and phenotypic level, between the two species. These differences include the presence of hybrids, variations in wing phenotype, and genomic patterns of introgression and differentiation. In I. salapia, the two lineages appear to hybridize only rarely, whereas in O. onega the hybrids are not only more common, but also genetically and phenotypically more variable. We also detected loci statistically associated with wing colour pattern variation, but in both species these loci were not over‐represented among the candidate barrier loci, suggesting that traits other than wing colour pattern may be important for reproductive isolation. Our results contrast with the genomic patterns observed between hybridizing lineages in the mimetic Heliconius butterflies, and call for a broader investigation into the genomics of speciation in Ithomiini ‐ the largest radiation of mimetic butterflies.     

Synthesis and biological activities of aminopyrimidyl-indoles structurally related to meridianins

The synthesis of new meridianin derivatives substituted at the C-5 position of the 2-aminopyrimidine ring by various aryl groups and substituted or not by a methyl group on the indole nitrogen is described. These compounds were tested for their kinase inhibitory potencies toward five kinases (CDK5/p25, CK1δ/ε, GSK-3α/β, Dyrk1A and Erk2) as well as their in vitro antiproliferative activities toward a human fibroblast primary culture and two human solid cancer cell lines (MCF-7 and PA 1).     

Morphological criteria to identify faecal pellets of sympatric ungulates in West African savanna and estimates of associated error

Indirect surveys may prove to be useful tools in complementing classical direct counts when monitoring ungulate populations and may also promote better understanding of the precise structure and functioning of the rich ungulate communities of African savannas. However, the identification of faecal pellets can be difficult where several sympatric species occur. This study develops simple field criteria for distinguishing between pellets among ten sympatric West African ungulates. A discriminant analysis was performed, using the mean of measurements of pellet groups from different species to pinpoint and characterize the most useful morphological criteria for separation between them. The mean diameter of pellets within each pellet group proved to be the most valuable variable for species segregation, whilst the second axis separated species by mean indent depth. The pellet groups of six of the ten designated species could be identified with a minimum misclassification error. However, no simple morphological variables emerged to permit discrimination between hartebeest and topi, or between bushbuck and Bohor reedbuck pellets. Once pellet groups have been identified, their density and spatial distribution may provide useful information on the use of space and habitat of sympatric species, over given periods.     

Rational enhancement of the coordination capability of Ru(III)(salen)-nitronyl nitroxide building block: A step towards 2p–3d–4d magnetic edifices

The reaction of [Ru(salen)(PPh₃)Cl] and the 5-imidazol-substituted nitronyl nitroxide radical (NIT-(5)ImH) yields the [Ru(salen)(PPh₃)(NIT-(5)ImH)](ClO₄) (1) complex which has been characterized by single crystal X-ray diffraction. This analysis reveals that the Ru(III) ion is coordinated to a tetradentate salen^2? ligand in equatorial positions while one PPh₃ ligand and one NIT-(5)ImH radical are coordinated in axial positions. This led to Ru^III ions in tetragonally elongated octahedral geometry. From the magnetic point of view ferromagnetic intramolecular interaction (J₁ = +2.47 cm^?1) have been found between the Ru(III) ion and the coordinated NIT-(5)ImH while no significant intermolecular antiferromagnetic interactions are observed at low temperature leading to a ground spin state S = 1. The absence of intermolecular magnetic interaction is explained by considering the crystal packing of (1) where the [Ru(salen)(PPh₃)(NIT-(5)ImH)]⁺ moieties are relatively well isolated. This has to be compared with the situation observed in the previously reported [Ru(salen)(PPh₃)-(NIT)]⁺ compound (2) where ferromagnetic Ru^III–NIT interaction were identified and the crystal packing generate intermolecular antiferromagnetic interactions that complicated the study. The analysis of this compound confirms the rather isotropic g values that were found of (2) and of [Ru(salen)(PPh₃)(N₃)], (3) a radical-free analogue. Moreover it is also a step towards extended structures based on Ru^III–NIT moieties since this compound possesses a free bischelating site likely to coordinate additional metallic ions.