Identification and functional characterization of the Arabidopsis Snf1‐related protein kinase SnRK2.4 phosphatidic acid‐binding domain

Phosphatidic acid (PA) is an important signalling lipid involved in various stress‐induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA‐binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA‐binding domain (PABD) of SnRK2.4. Unlike the full‐length SnRK2.4, neither the PABD‐YFP fusion protein nor the SnRK2.10 re‐localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re‐localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non‐PA‐binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA–SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development.     

Molecular genetics of preeclampsia and HELLP syndrome — A review

Preeclampsia is characterised by new onset hypertension and proteinuria and is a major obstetrical problem for both mother and foetus. Haemolysis elevated liver enzymes and low platelets (HELLP) syndrome is an obstetrical emergency and most cases occur in the presence of preeclampsia. Preeclampsia and HELLP are complicated syndromes with a wide variety in severity of clinical symptoms and gestational age at onset. The pathophysiology depends not only on periconceptional conditions and the foetal and placental genotype, but also on the capability of the maternal system to deal with pregnancy. Genetically, preeclampsia is a complex disorder and despite numerous efforts no clear mode of inheritance has been established. A minor fraction of HELLP cases is caused by foetal homozygous LCHAD deficiency, but for most cases the genetic background has not been elucidated yet. At least 178 genes have been described in relation to preeclampsia or HELLP syndrome. Confined placental mosaicism (CPM) is documented to cause early onset preeclampsia in some cases; the overall contribution of CPM to the occurrence of preeclampsia has not been adequately investigated yet. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.     

The Network of Knowledge approach: improving the science and society dialogue on biodiversity and ecosystem services in Europe

The absence of a good interface between scientific and other knowledge holders and decision-makers in the area of biodiversity and ecosystem services has been recognised for a long time. Despite recent advancements, e.g. with the Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES), challenges remain, particularly concerning the timely provision of consolidated views from different knowledge domains. To address this challenge, a strong and flexible networking approach is needed across knowledge domains and institutions. Here, we report on a broad consultation process across Europe to develop a Network of Knowledge on biodiversity and ecosystem services (NoK), an approach aiming at (1) organising institutions and knowledge holders in an adaptable and responsive framework and (2) informing decision-makers with timely and accurate biodiversity knowledge. The consultation provided a critical analysis of the needs that should be addressed by a NoK and how it could complement existing European initiatives and institutions at the interface between policy and science. Among other functions, the NoK provides consolidated scientific views on contested topics, identification of research gaps to support relevant policies, and horizon scanning activities to anticipate emerging issues. The NoK includes a capacity building component on interfacing activities and contains mechanisms to ensure its credibility, relevance and legitimacy. Such a network would need to ensure credibility, relevance and legitimacy of its work by maximizing transparency and flexibility of processes, quality of outputs, the link to data and knowledge provision, the motivation of experts for getting involved and sound communication and capacity building.     

Age-Related Longitudinal Changes in Metabolic Energy Expenditure during Walking in Boys with Duchenne Muscular Dystrophy

Objective

The aim of this study was to evaluate age-related changes in metabolic walking energy expenditure in ambulant boys affected by Duchenne muscular dystrophy over a follow-up period of 12 months.

Methods

At baseline (T1) and 12 months later (T2), metabolic walking energy expenditure was assessed during a 6-minute walk test at comfortable speed in 14 ambulant boys with Duchenne (age range: 6.0-12.5 years, mean 8.2). Outcome measures derived from the assessment included the 6-minute comfortable walking distance (m) and net-nondimensional energy cost relative to speed-matched control cost (SMC-EC, %). Statistical comparisons were made using a two-way repeated measures ANOVA (factors: time (T1 versus T2) and age (<8 years of age (yoa) versus ≥8 yoa)).

Results

Over the course of the study, a significant decrease of -28m (−8.2%, p = 0.043) was noted in the walked distance at comfortable speed. Besides, SMC-EC increased with 4.4%, although this change was not significant (p = 0.452). Regarding age groups, boys below 8 yoa showed a smaller annual decrease in the walked distance (−15 m) compared to boys above 8 yoa (−37 m). SMC-EC increased with 10% in the older boys, while in the younger boys it decreased (−2.1%). The main effect of age group on walking distance and SMC-EC however was not significant (p>0.158), and also there were no interaction effects (p>0.248).

Conclusions

The results of our small study suggest that the natural course of walking performance in ambulant boys with Duchenne is characterized by a decrease in comfortable walking distance and an increase in walking energy cost. The rate of energy cost seems to increase with age, while walking distance decreases, which is opposite from the trend in typically developing children.     

The cerebellum link to neuroticism: a volumetric MRI association study in healthy volunteers

Prior research suggests an association between reduced cerebellar volumes and symptoms of depression and anxiety in patients with mood disorders. However, whether a smaller volume in itself reflects a neuroanatomical correlate for increased susceptibility to develop mood disorders remains unclear. The aim of the present study was to examine the relationship between cerebellar volume and neurotic personality traits in a non-clinical subject sample. 3T Structural magnetic resonance imaging scans were acquired, and trait depression and anxiety scales of the revised NEO personality inventory were assessed in thirty-eight healthy right-handed volunteers. Results showed that cerebellar volume corrected for total brain volume was inversely associated with depressive and anxiety-related personality traits. Cerebellar gray and white matter contributed equally to the observed associations. Our findings extend earlier clinical observations by showing that cerebellar volume covaries with neurotic personality traits in healthy volunteers. The results may point towards a possible role of the cerebellum in the vulnerability to experience negative affect. In conclusion, cerebellar volumes may constitute a clinico-neuroanatomical correlate for the development of depression- and anxiety-related symptoms.     

Diet shift of a facultative scavenger, the wolverine, following recolonization of wolves

1. Wolves Canis lupus L. recolonized the boreal forests in the southern part of the Scandinavian peninsula during the late 1990s, but so far there has been little attention to its effect on ecosystem functioning. Wolf predation increases the availability of carcasses of large prey, especially moose Alces alces L., which may lead in turn to a diet switch in facultative scavengers such as the wolverine Gulo gulo L. 2. Using 459 wolverine scats collected during winter-spring 2001-04 for DNA identity and dietary contents, we compared diet inside and outside wolf territories while controlling for potential confounding factors, such as prey density. We tested the hypothesis that wolverine diet shifted towards moose in the presence of wolves, while taking into account possible sexual segregation between the sexes. Occurrence of reindeer, moose and small prey was modelled against explanatory covariates using logistic mixed-effects models. Furthermore, we compared diet composition and breadth among habitats and sexes. 3. Occurrence of reindeer, moose and small prey in the diet varied with prey availability and habitat. As expected, diet contained more moose and less reindeer and small prey in the presence of wolves. Their diet in tundra consisted of 40% reindeer Rangifer tarandus L., 39% moose and 9% rodents. In forest with wolf, their diet shifted to 76% moose, 18% reindeer and 5% rodents; compared to 42% moose, 32% reindeer and 15% rodents in forest without wolf. This diet switch could not be explained by higher moose density in wolf territories. Female diet consisted of more small prey than for males, but there was a tendency for females to use the highly available moose carrion opportunistically and to hunt less on small prey within wolf territories. 4. Our study highlights how wolves increase scavenging opportunities for wolverines, and how sexual differences in diet may also apply to large scavengers. Due to their more restricted home range, female wolverines are forced to rely more on hunting small prey. The relatively high occurrence of wolf kills, however, forms an important food source to wolverines in this area. The recolonization of wolves may therefore have contributed to the consequent recolonization of wolverines into the same area.     

Behavioural patterns in ewe–lamb pairs and vulnerability to predation by wolverines

In Norway domestic sheep Ovis aries range unattended in mountainous areas during the summer season. Wolverines Gulo gulo re-established in the alpine regions of southern Norway during recent decades and are viewed as a substantial predator on lambs. Reducing predation on sheep by wolverines would not only reduce the economic loss to farmers but also promote the acceptance of wolverines in their summer ranges. We hypothesized that male lambs would be more prone to wolverine predation, because of higher locomotor activity, lower behavioural ewe–lamb synchrony and larger ewe–lamb distance. We studied ewe and lamb behavioural patterns, synchrony and ewe–lamb distance on a summer range in Knutshøi, south-central Norway. Although no differences were found in ewe–lamb distance or locomotor activity in gender, female lambs synchronized their behaviour more with their mother than males. Only for female lambs, increased synchrony resulted in closer ewe–lamb distances. Overall losses to wolverines based on long-term data indicate that male lambs are more prone to predation than females later in the season. These sex-specific behavioural differences in lambs affect the spatial and social relationships between ewe and lamb, and are likely to increase with age eventually leading to sexual segregation. Male lambs can therefore be expected to be more prone to wolverine predation towards the end of the season, when lambs become independent from the ewe.     

The reversibility of the glutathionyl-quercetin adduct spreads oxidized quercetin-induced toxicity

Quercetin is one of the most prominent dietary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its o-quinone/quinone methide QQ. QQ is toxic since it instantaneously reacts with thiols of, e.g., proteins. In cells, QQ will initially form an adduct with glutathione (GSH), giving GSQ. We have found that GSQ is not stable; it dissociates continuously into GSH and QQ with a half life of 2min. Surprisingly, GSQ incubated with 2-mercapto-ethanol (MSH), a far less reactive thiol, results in the conversion of GSQ into the MSH-adduct MSQ. A similar conversion of GSQ into relatively stable protein thiol-quercetin adducts is expected. With the dithiol dihydrolipoic acid (L(SH)(2)), quercetin is formed out of GSQ. These results indicate that GSQ acts as transport and storage of QQ. In that way, the initially highly focussed toxicity of QQ is dispersed by the formation of GSQ that finally spreads QQ-induced toxicity, probably even over cells.     

Anti-cancer effects of vitamin C revisited

Vitamin C was first suggested to have cancer-fighting properties in the 1930s and has been the subject of controversy ever since. Despite repeated reports of selective cancer cell toxicity induced by high-dose vitamin C treatment in vitro and in mouse models, the mechanism of action has remained elusive.Yun et al.¹ have recently shed light on what was until now the elusive mechanism by which vitamin C (aka ascorbate) induces toxicity in selected oncogene-driven cancers. They reported that in cells with mutations of KRAS or BRAF, death is not caused by vitamin C itself, but rather by its oxidized form dehydroascorbate (DHA). Whereas vitamin C enters cells through sodium cotransporters, DHA competes with glucose for uptake through glucose transporters (particularly GLUT1 and GLUT4) and then is reduced back to vitamin C in cells² (Figure 1). It was previously observed that while melanoma cell lines take up DHA at much higher rates than vitamin C, normal melanocytes do not, demonstrating that transformation-driven upregulation of GLUTs leads to increased uptake of DHA³. More recently, using Magnetic Resonance Spectroscopy Imaging, it was demonstrated that hyperpolarized ¹³C-labeled DHA is rapidly taken up by cancer cells and converted to vitamin C, illustrating the tumors'' reducing state⁴. Yun et al.¹ now show that the reduction of DHA back to vitamin C is at the crux of the vitamin C-induced cell death observed in these cancer cells.Open in a separate windowFigure 1Mechanistic overview of proposed vitamin C toxicity in CRCs driven by KRAS and BRAF mutations. KRAS and BRAF mutations induce metabolic reprogramming by upregulating GLUT1, glucose uptake, and glycolytic flux. Upon vitamin C treatment and its extracellular oxidation, DHA (the oxidized form of vitamin C) is taken up through GLUT1 and is reduced back to vitamin C in the cells, depleting GSH and NADPH. Consequently, an increase in ROS leads to GAPDH oxidation, and with it, to a decrease in glycolytic flux. In parallel, ROS-mediated oxidative DNA damage induces PARP activation and subsequently, NAD⁺ levels fall and cause additional inhibition of GAPDH and glycolysis, resulting in energy crisis and cell death.Mutations in KRAS or BRAF are found in approximately half of the cases of colorectal cancer (CRC) and their expression correlates with an increase in GLUT1 expression, glucose uptake and reliance on glycolysis. Yun et al.¹ observed that vitamin C is oxidized to DHA in tissue culture media and that KRAS or BRAF mutated CRC cell lines take up more DHA compared to their wild-type counterparts. More importantly, they found that DHA induces death in the mutant lines, but not in wild-type counterparts overexpressing GLUT1, suggesting that additional oncogenic reprogramming is necessary for DHA-induced toxicity. The authors then profiled metabolic changes after treatment with vitamin C. In cells with KRAS or BRAF mutations, they found an accumulation of the glycolytic intermediates upstream of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) whereas those downstream of GAPDH were depleted. This indicated a decrease in GAPDH activity and a concomitant diversion of glucose into the oxidative phase of the pentose phosphate pathway (PPP), a metabolic shift that, upon oxidative stress, helps restore NADPH levels and cellular reducing potential (Figure 1). Indeed, Yun and co-workers found that the intracellular reduction of DHA back to vitamin C depleted the cellular stores of reduced glutathione (GSH, the major antioxidant in cells), leading to an increase in reactive oxygen species (ROS). Furthermore, they found that, upon exposure to DHA, GAPDH itself was oxidized (on Cys¹⁵²), and consequently inhibited. Inhibition of GAPDH caused energy stress in the highly glycolytic mutant cell lines, leading to cell death. In mice harboring tumor xenografts with either KRAS or BRAF mutations, treatment with high doses of vitamin C reduced tumor size. Treatment also reduced the number and size of polyps in an Apc-driven transgenic mouse model of intestinal cancer, but again, only in tumors expressing mutant Kras. Moreover, in addition to showing the direct inhibition of GAPDH by oxidation, the authors demonstrated that its activity is further hindered by DHA-induced NAD⁺ depletion, since GAPDH activity relies on the availability of NAD⁺ as a co-substrate. The major NAD⁺ consumer, the DNA repairing enzyme poly ADP-ribose polymerase (PARP) was then investigated. It was found that the increase in ROS after high-dose vitamin C treatment also induced DNA damage and PARP activation in KRAS or BRAF transformed cells. Providing the cells with a PARP inhibitor or an NAD⁺ precursor partially rescued their viability. Thus, ROS cause the inhibition of GAPDH activity in cells on two fronts: first, via inducing its direct oxidation and second, by causing NAD⁺ depletion (Figure 1). Yun et al.¹ thus demonstrated an intricate mechanism by which oncogenic reprogramming, which causes glycolysis addiction, induces a metabolic vulnerability which can be exploited with high doses of DHA that elevate intracellular ROS as it is converted back to vitamin C.Despite numerous clinical studies, the anti-cancer property of vitamin C has remained controversial. Potential translation of the mechanism presented by Yun et al.¹ to therapeutic application raises concerns regarding toxicities of high-dose vitamin C treatment. Though the authors do not report side effects in their mice (treated daily with 4-8 g/kg body weight IP), the upper dose equates to over half a kilogram for the average person. High-dose oral supplementation of vitamin C is associated with increased kidney stone incidence, and clinical studies demonstrated significant renal, cardiac, and metabolic toxicity upon vitamin C administration. Still, overall reports of toxicity are variable, poorly graded, and therefore inconclusive^5,6. Affinity studies of DHA for GLUT1 may help establish a lower effective dose, though unwanted side effects in tissues that highly express GLUT1 need to be considered. The brain obtains vitamin C by uptake of DHA through GLUT1 at the blood-brain barrier and its subsequent reduction⁷. Erythrocytes express high levels of GLUT1 and are crucial for ascorbate recycling, keeping DHA levels low⁸. Importantly, erythrocytes rely solely on glycolysis for energy production. Thus, high DHA levels may induce brain toxicity and haemolysis via mechanisms similar to those described by Yun et al.¹.Though vitamin C oxidizes rapidly in tissue culture media, it acts mainly as an antioxidant in vivo. It remains unclear how and where circulating vitamin C is oxidized in vivo, an issue not addressed by Yun et al.¹. Oxidation of vitamin C to DHA by tumor stroma has been suggested⁹, complicating the ability to predict tumor responsiveness to the treatment. As such, without being able to control the extent of vitamin C oxidation to DHA, effectiveness and toxicity of vitamin C treatment cannot be predicted.Finally, the authors report that, following DHA uptake, NAD⁺ depletion by PARP activation contributes to the inhibition of glycolysis (and potentially to the stimulation of oxidative PPP flux). The observation that PARP inhibition partly rescues vitamin C-treated cells may suggest that the toxic effect of DHA uptake is not caused by ROS alone, since restoring NAD⁺ levels and glycolysis with the PARP inhibitor may actually decrease PPP flux and NADPH production, and aggravate the redox stress. It remains to be demonstrated that PARP inhibition indeed restores NAD⁺ levels in vitamin C-treated cells, and how this affects the balance between energy and redox metabolism. This also raises the question whether PARP activity in BRCA1/2-deficient tumors produces a similar metabolic phenotype via NAD⁺ depletion and whether the use of a PARP inhibitor (e.g., olaparib) to treat these tumors¹⁰ might restore NAD⁺ levels and counterbalance GAPDH inhibition by other oxidative agents.In summary, Yun et al.¹ show that, in glycolysis-addicted KRAS and BRAF driven cancer cells, high-dose vitamin C treatment induces cell death via the uptake and reduction of its oxidized form DHA back to vitamin C. DHA reduction, through scavenging GSH, induces oxidative stress, leading to GAPDH inactivation, inhibition of glycolysis and the subsequent energy crisis and cell death. This study further elucidates the mechanism by which ROS can induce cell death, and neatly shows how vitamin C, an antioxidant, can work as a double edged sword. However, further work is necessary to determine whether there is a therapeutic potential for vitamin C in cancer patients.     

The Influence of Sex Steroids on Structural Brain Maturation in Adolescence

Puberty reflects a period of hormonal changes, physical maturation and structural brain reorganization. However, little attention has been paid to what extent sex steroids and pituitary hormones are associated with the refinement of brain maturation across adolescent development. Here we used high-resolution structural MRI scans from 215 typically developing individuals between ages 8–25, to examine the association between cortical thickness, surface area and (sub)cortical brain volumes with luteinizing hormone, testosterone and estradiol, and pubertal stage based on self-reports. Our results indicate sex-specific differences in testosterone related influences on gray matter volumes of the anterior cingulate cortex after controlling for age effects. No significant associations between subcortical structures and sex hormones were found. Pubertal stage was not a stronger predictor than chronological age for brain anatomical differences. Our findings indicate that sex steroids are associated with cerebral gray matter morphology in a sex specific manner. These hormonal and morphological differences may explain in part differences in brain development between boys and girls.