Modulation of abscisic acid signaling in vivo by an engineered receptor-insensitive protein phosphatase type 2C allele

The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1(W385A), which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1(W385A) caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1(W385A) qualifies as a hypermorphic mutation. Expression of hab1(W385A) in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.     

Macrophages from lupus-prone MRL mice have a conditional signaling abnormality that leads to dysregulated expression of numerous genes

Macrophages (mϕ) from pre-diseased mice of the major murine inbred models of spontaneous autoimmunity (AI), including multiple lupus-prone strains and the type I diabetes-prone NOD (non-obese diabetic) strain, have identical apoptotic target-dependent abnormalities. This characteristic feature of mϕ from AI-prone mice suggests that abnormal signaling events induced within mϕ following their interaction with apoptotic targets may predispose to AI. Such signaling abnormalities would affect predominantly the processing and presentation of self-antigen (i.e., derived from apoptotic targets), while sparing the processing and presentation of foreign antigen (i.e., derived from non-apoptotic sources). Here, we used DNA microarrays to test the hypothesis that mϕ from AI-prone mice (MRL/MpJ [MRL/+] or MRL/MpJ-Tnfrsf6 ^lpr [MRL/lpr]) differentially express multiple genes in comparison to non-AI mϕ (BALB/c), but do so in a largely apoptotic cell-dependent manner. Mϕ were stimulated with lipopolysaccharide, a potent innate stimulus, in the presence or absence of serum (an experimental surrogate for apoptotic targets). In accord with our hypothesis, the number of genes differentially expressed by MRL mϕ was significantly increased in the presence vs. the absence of serum, the apoptotic target surrogate (n = 401 vs. n = 201). Notably, for genes differentially expressed by MRL mϕ in the presence of serum, serum-free culture normalized their expression to a level statistically indistinguishable from that by non-AI mϕ. Comparisons of mϕ from AI-prone NOD and non-AI C57BL/6 mice corroborated these findings. Together, these data support the hypothesis that mϕ from MRL and other AI-prone mice are characterized by a conditional abnormality elicited by serum lipids or apoptotic targets.     

Factors affecting otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) abundance and breeding success in freshwater habitats of the northeastern Iberian Peninsula

Otters are elusive semi-aquatic mammals, occurring in low densities and difficult to count. A study was conducted in 15 stretches (10–12 km) of four tributaries of the Ebro River, with the aims to understand spatial and temporal changes in otter abundance and breeding success and the causes of such variations. Between 1990 and 2009, both otter parameters were assessed by means of visual spring–early summer censuses, carried out by sets of observers located every 500 m. A total of 134 censuses were carried out, involving 4,540 twilight watches (7,896.5 h) and 520 otter sightings were recorded. Also, 39 different habitat characteristics (including prey species) were established for each stretch. Otter abundance was assessed by means of three indexes related to the census effort and the length and surface units (in terms of otter habitat). Excluding the upper stretches, we found on average 0.07–0.26 adult and subadults per kilometre (1.45–6.26/km²). Important interannual fluctuations in otter abundance and breeding success were found in some Pyrenean stretches located downstream of a large reservoir. This was linked to the effect of autumn floods (in part due to the opening of dams) on the fish stocks (mainly Barbels). Similar habitat conditions resulted in similar abundances between stretches, and different habitat conditions generated different abundances in adjacent stretches of the same river. No significant differences were found for litter size between small cubs (1.60 ± 0.70 cubs per litter) and large cubs (1.39 ± 0.50) as a whole, but differences between stretches were found. On average we found between 0.009 and 0.130 large cubs per year per kilometre. Otters bred more successfully in stretches with higher otter densities. Otter abundance and breeding success correlated negatively with altitude as a consequence of the interaction of several habitat characteristics with altitude. The stepwise linear regression linked the number of adult otters per kilometre with the ecosystem production (chlorophyll a concentration), while the number of large cubs per kilometre per year was linked with the food (fish + crayfish) abundance. The findings bring about a consistent and congruent scenario of otter abundance and breeding success explained by the different steps along the food chain.     

Circulating omentin as a novel biomarker of endothelial dysfunction

Omentin is a novel soluble lectin expressed mainly in the stromal‐vascular cells from visceral adipose tissue with vasodilator effect in isolated blood vessels. To gain insight in the relationship between obesity and cardiovascular risk factors, we aimed to explore the interaction among circulating omentin, metabolic parameters, and endothelial function. Circulating omentin (enzyme‐linked immunosorbent assay) was studied in 248 white men (148 with normal glucose tolerance (NGT) and 100 with impaired glucose tolerance (IGT)). Insulin sensitivity was measured using the frequently sampled intravenous glucose tolerance test. Vascular reactivity was measured by high‐resolution ultrasound of the brachial artery. Circulating omentin concentration was significantly increased in lean compared with overweight and obese subjects (53.7 ± 16.9 vs. 45.2 ± 16.8 and vs. 40.1 ± 15.5 ng/ml, P < 0.0001). Circulating omentin concentration correlated with age, BMI, waist‐to‐hip ratio (WHR), percentage of fat mass, systolic and diastolic blood pressure, endothelium‐dependent and independent vasodilation (EDV and EIV), C‐reactive protein, and interleukin‐6 (IL‐6). In IGT subjects, circulating omentin concentration also correlated with insulin sensitivity, although this association did not remain significant after controlling for BMI. In a multiple linear regression analysis, circulating omentin concentration (P = 0.01), systolic blood pressure (P = 0.04), and BMI (P = 0.04) contributed independently to EDV after controlling for age and C‐reactive protein in IGT subjects. In NGT subjects, only circulating omentin concentration (P = 0.01) was significantly associated with EDV. In conclusion, circulating omentin concentration could be a useful marker of endothelial function.     

Misleading population estimates: biases and consistency of visual surveys and matrix modelling in the endangered bearded vulture

Conservation strategies for long-lived vertebrates require accurate estimates of parameters relative to the populations' size, numbers of non-breeding individuals (the "cryptic" fraction of the population) and the age structure. Frequently, visual survey techniques are used to make these estimates but the accuracy of these approaches is questionable, mainly because of the existence of numerous potential biases. Here we compare data on population trends and age structure in a bearded vulture (Gypaetus barbatus) population from visual surveys performed at supplementary feeding stations with data derived from population matrix-modelling approximations. Our results suggest that visual surveys overestimate the number of immature (<2 years old) birds, whereas subadults (3-5 y.o.) and adults (>6 y.o.) were underestimated in comparison with the predictions of a population model using a stable-age distribution. In addition, we found that visual surveys did not provide conclusive information on true variations in the size of the focal population. Our results suggest that although long-term studies (i.e. population matrix modelling based on capture-recapture procedures) are a more time-consuming method, they provide more reliable and robust estimates of population parameters needed in designing and applying conservation strategies. The findings shown here are likely transferable to the management and conservation of other long-lived vertebrate populations that share similar life-history traits and ecological requirements.     

Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway

Background

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF^V600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.

Methodology/Principal Findings

The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.

Conclusions/Significance

Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.     

Metabolomics reveals reduction of metabolic oxidation in women with polycystic ovary syndrome after pioglitazone-flutamide-metformin polytherapy

Polycystic ovary syndrome (PCOS) is a variable disorder characterized by a broad spectrum of anomalies, including hyperandrogenemia, insulin resistance, dyslipidemia, body adiposity, low-grade inflammation and increased cardiovascular disease risks. Recently, a new polytherapy consisting of low-dose flutamide, metformin and pioglitazone in combination with an estro-progestagen resulted in the regulation of endocrine clinical markers in young and non-obese PCOS women. However, the metabolic processes involved in this phenotypic amelioration remain unidentified. In this work, we used NMR and MS-based untargeted metabolomics to study serum samples of young non-obese PCOS women prior to and at the end of a 30 months polytherapy receiving low-dose flutamide, metformin and pioglitazone in combination with an estro-progestagen. Our results reveal that the treatment decreased the levels of oxidized LDL particles in serum, as well as downstream metabolic oxidation products of LDL particles such as 9- and 13-HODE, azelaic acid and glutaric acid. In contrast, the radiuses of small dense LDL and large HDL particles were substantially increased after the treatment. Clinical and endocrine-metabolic markers were also monitored, showing that the level of HDL cholesterol was increased after the treatment, whereas the level of androgens and the carotid intima-media thickness were reduced. Significantly, the abundance of azelaic acid and the carotid intima-media thickness resulted in a high degree of correlation. Altogether, our results reveal that this new polytherapy markedly reverts the oxidant status of untreated PCOS women, and potentially improves the pro-atherosclerosis condition in these patients.     

Transketolase-like 1 expression is modulated during colorectal cancer progression and metastasis formation

Background

Transketolase-like 1 (TKTL1) induces glucose degradation through anaerobic pathways, even in presence of oxygen, favoring the malignant aerobic glycolytic phenotype characteristic of tumor cells. As TKTL1 appears to be a valid biomarker for cancer prognosis, the aim of the current study was to correlate its expression with tumor stage, probability of tumor recurrence and survival, in a series of colorectal cancer patients.

Methodolody/Principal Findings

Tumor tissues from 63 patients diagnosed with colorectal cancer at different stages of progression were analyzed for TKTL1 by immunohistochemistry. Staining was quantified by computational image analysis, and correlations between enzyme expression, local growth, lymph-node involvement and metastasis were assessed. The highest values for TKTL1 expression were detected in the group of stage III tumors, which showed significant differences from the other groups (Kruskal-Wallis test, P = 0.000008). Deeper analyses of T, N and M classifications revealed a weak correlation between local tumor growth and enzyme expression (Mann-Whitney test, P = 0.029), a significant association of the enzyme expression with lymph-node involvement (Mann-Whitney test, P = 0.0014) and a significant decrease in TKTL1 expression associated with metastasis (Mann-Whitney test, P = 0.0004).

Conclusions/Significance

To our knowledge, few studies have explored the association between variations in TKTL1 expression in the primary tumor and metastasis formation. Here we report downregulation of enzyme expression when metastasis appears, and a correlation between enzyme expression and regional lymph-node involvement in colon cancer. This finding may improve our understanding of metastasis and lead to new and more efficient therapies against cancer.