Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

Root fungal endophytes: identity,phylogeny and roles in plant tolerance to metal stress

Metal trace elements accumulate in soils mainly because of anthropic activities, leading living organisms to develop strategies to handle metal toxicity. Plants often associate with root endophytic fungi, including nonmycorrhizal fungi, and some of these organisms are associated with metal tolerance. The lack of synthetic analyses of plant-endophyte-metal tripartite systems and the scant consideration for taxonomy led to this review aiming (1) to inventory non-mycorrhizal root fungal endophytes described with respect to their taxonomic diversity and (2) to determine the mutualistic roles of these plant-fungus associations under metal stress. More than 1500 species in 100 orders (mainly Hypocreales and Pleosporales) were reported from a wide variety of environments and hosts. Most reported endophytes had a positive effect on their host under metal stress, but with various effects on metal uptake or translocation and no clear taxonomic consistency. Future research considering the functional patterns and dynamics of these associations is thus encouraged.     

Inhibitory effects of estrogens on digestive enzymes,insulin deficiency,and pancreas toxicity in diabetic rats

Diabetes mellitus, with its attendant disorders and dysfunctional behaviors, constitutes a growing concern to the population of the world. With this concern in mind, the present study investigated the anti-diabetic and hypolipedimic potential of 17β-estradiol (called E2), particularly in terms of its inhibitory effects on maltase, sucrase, lactase, and lipase activities in the intestine of surviving diabetic rats. The findings revealed that this supplement helped protect the β cells of the rats from death and damage. Interestingly, E2 induced considerable decreases of 29%, 46%, 42%, and 84% in the activities of intestinal maltase, lactase, sucrase, and lipase, respectively. The E2 extract also decreased the glucose, triglyceride, and total cholesterol rates in the plasma of diabetic rats by 39%, 27%, and 53%, respectively, and increased the HDL–cholesterol level by 74%, which helped maintain the homeostasis of blood lipid. When compared to those of the untreated diabetic rats, the superoxide dismutase, catalase, and glutathione peroxidase levels in the pancreas of the rats treated with this supplement were also enhanced by 330%, 170%, and 301%, respectively. A significant decrease was also observed in the lipid peroxidation level and lactate dehydrogenase activity in the pancreas of diabetic rats after E2 administration. Overall, the findings presented in this study demonstrate that E2 has both a promising potential with regard to the inhibition of intestinal maltase, sucrase, lactase, and lipase activities, and a valuable hypoglycemic and hypolipidemic function, which make it a potential strong candidate for industrial application as apharmacological agent for the treatment and prevention of hyperlipidemia, obesity, and cardiovascular diseases.     

Detecting the footprint of changing atmospheric nitrogen deposition loads on acid grasslands in the context of climate change

Although atmospheric nitrogen (N) deposition and climate changes are both recognized as major components of global change, their interaction at ecosystem level is less well understood. A stratified resampling approach was used to investigate the potential impact of changing levels of atmospheric nitrogen deposition and climate change on species composition of nutrient‐poor acid grasslands within the French Atlantic Domain (FAD). The study was based on a comparison, over a period of 25 years, of 162 past and present vegetation records assigned to the species‐rich Nardus grasslands and distributed in regional community types (CTs). Similarly, the characterization of N deposition and climate was stratified according to (i) past (1980–1990) and present (1995–2005) periods, and (ii) FAD and CT scales. Despite the relatively short time span between sampling periods, significant N deposition and climate changes were detected as well as vegetation changes. Correspondence analysis showed that the relative importance of N deposition and climate in explaining vegetation changes depended on the spatial scale of investigation (FAD vs. local CTs) and the CT. At the FAD scale, the increase of annual mean temperature and decrease of water availability were clearly related to the changes in floristic composition. At the local scale, the most stable CT experienced no significant climate change and a stable load of N deposition, whereas the CTs characterized by the largest floristic changes were associated with dramatic climate changes and moderate loads in both oxidized and reduced N deposition. Despite the narrow gradient of deposition investigated, N deposition was related to significant grassland community changes, depending on the region, i.e. climate context, and on whether N deposition was in the oxidized or reduced form. Our results suggest that N deposition drives grassland composition at the local scale, in interaction with climate, whereas climate changes remain the predominant driver at the FAD scale.     

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.     

Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor

The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R??????? fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2???????, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a 1?N-13C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.     

Atorvastatin prevents <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> cytoadherence and endothelial damage

Background

The adhesion of Plasmodium falciparum parasitized red blood cell (PRBC) to human endothelial cells (EC) induces inflammatory processes, coagulation cascades, oxidative stress and apoptosis. These pathological processes are suspected to be responsible for the blood-brain-barrier and other organs' endothelial dysfunctions observed in fatal cases of malaria. Atorvastatin, a drug that belongs to the lowering cholesterol molecule family of statins, has been shown to ameliorate endothelial functions and is widely used in patients with cardiovascular disorders.

Methods

The effect of this compound on PRBC induced endothelial impairments was assessed using endothelial co-culture models.

Results

Atorvastatin pre-treatment of EC was found to reduce the expression of adhesion molecules and P. falciparum cytoadherence, to protect cells against PRBC-induced apoptosis and to enhance endothelial monolayer integrity during co-incubation with parasites.

Conclusions

These results might suggest a potential interest use of atorvastatin as a protective treatment to interfere with the pathophysiological cascades leading to severe malaria.

     

Cost-effectiveness of early versus standard antiretroviral therapy in HIV-infected adults in Haiti

Background

In a randomized clinical trial of early versus standard antiretroviral therapy (ART) in HIV-infected adults with a CD4 cell count between 200 and 350 cells/mm³ in Haiti, early ART decreased mortality by 75%. We assessed the cost-effectiveness of early versus standard ART in this trial.

Methods and Findings

Trial data included use of ART and other medications, laboratory tests, outpatient visits, radiographic studies, procedures, and hospital services. Medication, laboratory, radiograph, labor, and overhead costs were from the study clinic, and hospital and procedure costs were from local providers. We evaluated cost per year of life saved (YLS), including patient and caregiver costs, with a median of 21 months and maximum of 36 months of follow-up, and with costs and life expectancy discounted at 3% per annum. Between 2005 and 2008, 816 participants were enrolled and followed for a median of 21 months. Mean total costs per patient during the trial were US$1,381 for early ART and US$1,033 for standard ART. After excluding research-related laboratory tests without clinical benefit, costs were US$1,158 (early ART) and US$979 (standard ART). Early ART patients had higher mean costs for ART (US$398 versus US$81) but lower costs for non-ART medications, CD4 cell counts, clinically indicated tests, and radiographs (US$275 versus US$384). The cost-effectiveness ratio after a maximum of 3 years for early versus standard ART was US$3,975/YLS (95% CI US$2,129/YLS–US$9,979/YLS) including research-related tests, and US$2,050/YLS excluding research-related tests (95% CI US$722/YLS–US$5,537/YLS).

Conclusions

Initiating ART in HIV-infected adults with a CD4 cell count between 200 and 350 cells/mm³ in Haiti, consistent with World Health Organization advice, was cost-effective (US$/YLS <3 times gross domestic product per capita) after a maximum of 3 years, after excluding research-related laboratory tests.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00120510","term_id":"NCT00120510"}}NCT00120510 Please see later in the article for the Editors'' Summary