Oestradiol and osteoclast differentiation: Effects on p53 and mitochondrial metabolism.

BACKGROUND: Oestrogen deficiency increases bone resorption, contributing to osteoporosis development. Yet, the mechanisms mediating the effects of oestrogen on osteoclasts remain unclear. This study aimed to elucidate the early metabolic alteration induced by RANKL, the essential cytokine in osteoclastogenesis and 17-beta-oestradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and primary bone marrow-derived macrophages as biological models. RESULTS: This research demonstrated that, in osteoclast precursors, RANKL stimulates complex I activity, oxidative phosphorylation (OXPHOS) and mitochondria-derived ATP production as early as 3 h of exposure. This effect on mitochondrial bioenergetics is associated with an increased capacity to oxidize TCA cycle substrates, fatty acids and amino acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3 h. Further, the pro-apoptotic effects of E2 during osteoclast differentiation were associated with an accumulation of p392S-p53 in mitochondria. CONCLUSIONS: These findings elucidate the early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis.

Quantitative analysis of neuronal mitochondrial movement reveals patterns resulting from neurotoxicity of rotenone and 6-hydroxydopamine.

Alterations in mitochondrial dynamics, including their intracellular trafficking, are common early manifestations of neuronal degeneration. However, current methodologies used to study mitochondrial trafficking events rely on parameters that are primarily altered in later stages of neurodegeneration. Our objective was to establish a reliable applied statistical analysis to detect early alterations in neuronal mitochondrial trafficking. We propose a novel quantitative analysis of mitochondria trajectories based on innovative movement descriptors, including straightness, efficiency, anisotropy, and kurtosis. We evaluated time- and dose-dependent alterations in trajectory descriptors using biological data from differentiated SH-SY5Y cells treated with the mitochondrial toxicants 6-hydroxydopamine and rotenone. MitoTracker Red CMXRos-labelled mitochondria movement was analyzed by total internal reflection fluorescence microscopy followed by computational modelling to describe the process. Based on the aforementioned trajectory descriptors, this innovative analysis of mitochondria trajectories provides insights into mitochondrial movement characteristics and can be a consistent and sensitive method to detect alterations in mitochondrial trafficking occurring in the earliest time points of neurodegeneration.

Evaluation of 6-Hydroxydopamine and Rotenone In Vitro Neurotoxicity on Differentiated SH-SY5Y Cells Using Applied Computational Statistics.

With the increase in life expectancy and consequent aging of the world's population, the prevalence of many neurodegenerative diseases is increasing, without concomitant improvement in diagnostics and therapeutics. These diseases share neuropathological hallmarks, including mitochondrial dysfunction. In fact, as mitochondrial alterations appear prior to neuronal cell death at an early phase of a disease's onset, the study and modulation of mitochondrial alterations have emerged as promising strategies to predict and prevent neurotoxicity and neuronal cell death before the onset of cell viability alterations. In this work, differentiated SH-SY5Y cells were treated with the mitochondrial-targeted neurotoxicants 6-hydroxydopamine and rotenone. These compounds were used at different concentrations and for different time points to understand the similarities and differences in their mechanisms of action. To accomplish this, data on mitochondrial parameters were acquired and analyzed using unsupervised (hierarchical clustering) and supervised (decision tree) machine learning methods. Both biochemical and computational analyses resulted in an evident distinction between the neurotoxic effects of 6-hydroxydopamine and rotenone, specifically for the highest concentrations of both compounds.

Correction: Solvation of Li+ by argon: how important are three-body forces?

Correction for 'Solvation of Li+ by argon: how important are three-body forces?' by Frederico V. Prudente et al., Phys. Chem. Chem. Phys., 2017, 19, 25707-25716.

Solvation of Li+ by argon: how important are three-body forces?

A new analytical potential for Li+Ar2 including three-body interactions has been modeled by employing ab initio energies that were calculated within the CCSD(T) framework and a quadruple-zeta basis-set (i.e., cc-pVQZ for lithium and aug-cc-pVQZ for argon) and, then, corrected for the basis-set superposition error (BSSE) with the counterpoise method. Departing from this function, we have constructed the potential energy surface for Li+Arn clusters by summing over all two-body and three-body terms. We have employed our evolutionary algorithm (EA) to perform a global geometry optimization that allows for the study of a Li+ ion microsolvated with argon atoms. For the smaller clusters, the putative global minimum geometry obtained for the analytical potential has been used as a starting point for an ab initio optimization at the MP2 level. For clusters up to n = 10, the energetics and structures from the analytical potential energy surface (PES) that includes three-body interactions show good agreement with the corresponding ones optimized at the ab initio level. Removing the three-body terms from the analytical PES leads to global minima that fail to represent the main energetic features and the structures become wrong in the case of the Li+Ar2, Li+Ar3 and Li+Ar10 clusters. For n > 10, the comparison between potentials with and without three-body forces shows significant structural and energetic differences for most of the cluster sizes.

Heuristics for multiobjective multiple sequence alignment.

BACKGROUND: Aligning multiple sequences arises in many tasks in Bioinformatics. However, the alignments produced by the current software packages are highly dependent on the parameters setting, such as the relative importance of opening gaps with respect to the increase of similarity. Choosing only one parameter setting may provide an undesirable bias in further steps of the analysis and give too simplistic interpretations. In this work, we reformulate multiple sequence alignment from a multiobjective point of view. The goal is to generate several sequence alignments that represent a trade-off between maximizing the substitution score and minimizing the number of indels/gaps in the sum-of-pairs score function. This trade-off gives to the practitioner further information about the similarity of the sequences, from which she could analyse and choose the most plausible alignment. METHODS: We introduce several heuristic approaches, based on local search procedures, that compute a set of sequence alignments, which are representative of the trade-off between the two objectives (substitution score and indels). Several algorithm design options are discussed and analysed, with particular emphasis on the influence of the starting alignment and neighborhood search definitions on the overall performance. A perturbation technique is proposed to improve the local search, which provides a wide range of high-quality alignments. RESULTS AND CONCLUSIONS: The proposed approach is tested experimentally on a wide range of instances. We performed several experiments with sequences obtained from the benchmark database BAliBASE 3.0. To evaluate the quality of the results, we calculate the hypervolume indicator of the set of score vectors returned by the algorithms. The results obtained allow us to identify reasonably good choices of parameters for our approach. Further, we compared our method in terms of correctly aligned pairs ratio and columns correctly aligned ratio with respect to reference alignments. Experimental results show that our approaches can obtain better results than TCoffee and Clustal Omega in terms of the first ratio.

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates.

The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.

A detailed investigation on the global minimum structures of mixed rare-gas clusters: geometry, energetics, and site occupancy.

We performed a global minimum search of mixed rare-gas clusters by applying an evolutionary algorithm (EA), which was recently proposed for binary atomic systems (Marques and Pereira, Chem. Phys. Lett. 2010, 485, 211). Before being applied to the potentials used in this work, the EA was further tested against results previously reported for the Ar(N)Xe(38-N) clusters and several new putative global minima were discovered. We employed either simple Lennard-Jones (LJ) potentials or more realistic functions to describe pair interactions in Ar(N)Kr(38-N), Ar(N)Xe(38-N), and Kr(N)Xe(38-N) clusters. The long-range tail of the pair-potentials shows some influence on the energetic features and shape of the structure of clusters. In turn, core-shell type structures are mostly observed for global minima of the binary rare-gas clusters, for both accurate and LJ potentials. However, the long-range tail of the potential may have influence on the type of atoms that segregate on the surface or form the core of the cluster. While relevant differences for the preferential site occupancy occur between the two potentials for Ar(N)Kr(38-N) (for N > 21), the type of atoms that segregate on the surface for Ar(N)Xe(38-N) and Kr(N)Xe(38-N) clusters is unaffected by the accuracy of the long-range part of the interaction in almost all cases. Moreover, the global minimum search for model-potentials in binary systems reveals that the surface-site occupancy is mainly determined by the combination of two parameters: the size ratio of the two types of particles forming the cluster and the minimum-energy ratio corresponding to the pair-interactions between unlike atoms.

Cell recycling during repeated very high gravity bio-ethanol fermentations using the industrial Saccharomyces cerevisiae strain PE-2.

A very high gravity (VHG) repeated-batch fermentation system using an industrial strain of Saccharomyces cerevisiae PE-2 (isolated from sugarcane-to-ethanol distillery in Brazil) and mimicking industrially relevant conditions (high inoculation rates and low O(2) availability) was successfully operated during fifteen consecutive fermentation cycles, attaining ethanol at 17.1 ± 0.2% (v/v) with a batch productivity of 3.5 ± 0.04 g l(-1) h(-1). Moreover, this innovative operational strategy (biomass refreshing step) prevented critical decreases on yeast viability levels and promoted high accumulation of intracellular glycerol and trehalose, which can provide an adaptive advantage to yeast cells under harsh industrial environments. This study contributes to the improvement of VHG fermentation processes by exploring an innovative operational strategy that allows attaining very high ethanol titres without a critical decrease of the viability level thus minimizing the production costs due to energy savings during the distillation process.

Integrative Profiling of Amyotrophic Lateral Sclerosis Lymphoblasts Identifies Unique Metabolic and Mitochondrial Disease Fingerprints.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with a rapid progression and no effective treatment. Metabolic and mitochondrial alterations in peripheral tissues of ALS patients may present diagnostic and therapeutic interest. We aimed to identify mitochondrial fingerprints in lymphoblast from ALS patients harboring SOD1 mutations (mutSOD1) or with unidentified mutations (undSOD1), compared with age-/sex-matched controls. Three groups of lymphoblasts, from mutSOD1 or undSOD1 ALS patients and age-/sex-matched controls, were obtained from Coriell Biobank and divided into 3 age-/sex-matched cohorts. Mitochondria-associated metabolic pathways were analyzed using Seahorse MitoStress and ATP Rate assays, complemented with metabolic phenotype microarrays, metabolite levels, gene expression, and protein expression and activity. Pooled (all cohorts) and paired (intra-cohort) analyses were performed by using bioinformatic tools, and the features with higher information gain values were selected and used for principal component analysis and Naïve Bayes classification. Considering the group as a target, the features that contributed to better segregation of control, undSOD1, and mutSOD1 were found to be the protein levels of Tfam and glycolytic ATP production rate. Metabolic phenotypic profiles in lymphoblasts from ALS patients with mutSOD1 and undSOD1 revealed unique age-dependent different substrate oxidation profiles. For most parameters, different patterns of variation in experimental endpoints in lymphoblasts were found between cohorts, which may be due to the age or sex of the donor. In the present work, we investigated several metabolic and mitochondrial hallmarks in lymphoblasts from each donor, and although a high heterogeneity of results was found, we identified specific metabolic and mitochondrial fingerprints, especially protein levels of Tfam and glycolytic ATP production rate, that may have a diagnostic and therapeutic interest.

Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells.

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function.

Selection of Saccharomyces cerevisiae strains for efficient very high gravity bio-ethanol fermentation processes.

An optimized very high gravity (VHG) glucose medium supplemented with low cost nutrient sources was used to evaluate bio-ethanol production by 11 Saccharomyces cerevisiae strains. The industrial strains PE-2 and CA1185 exhibited the best overall fermentation performance, producing an ethanol titre of 19.2% (v/v) corresponding to a batch productivity of 2.5 g l(-1) h(-1), while the best laboratory strain (CEN.PK 113-7D) produced 17.5% (v/v) ethanol with a productivity of 1.7 g l(-1) h(-1). The results presented here emphasize the biodiversity found within S. cerevisiae species and that naturally adapted strains, such as PE-2 and CA1185, are likely to play a key role in facilitating the transition from laboratory technological breakthroughs to industrial-scale bio-ethanol fermentations.

Mitochondrial remodeling in human skin fibroblasts from sporadic male Parkinson's disease patients uncovers metabolic and mitochondrial bioenergetic defects.

Parkinson's Disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra. The exact mechanism by which dopaminergic neurodegeneration occurs is still unknown; however, mitochondrial dysfunction has long been implicated in PD pathogenesis. To investigate the sub-cellular events that lead to disease progression and to develop personalized interventions, non-neuronal cells which are collected in a minimally invasive manner can be key to test interventions aimed at improving mitochondrial function. We used human skin fibroblasts from sporadic PD (sPD) patients as a cell proxy to detect metabolic and mitochondrial alterations which would also exist in a non-neuronal cell type. In this model, we used a glucose-free/galactose- glutamine- and pyruvate-containing cell culture medium, which forces cells to be more dependent on oxidative phosphorylation (OXPHOS) for energy production, in order to reveal hidden metabolic and mitochondrial alterations present in fibroblasts from sPD patients. We demonstrated that fibroblasts from sPD patients show hyperpolarized and elongated mitochondrial networks and higher mitochondrial ROS concentration, as well as decreased ATP levels and glycolysis-related ECAR. Our results also showed that abnormalities of fibroblasts from sPD patients became more evident when stimulating OXPHOS. Under these culture conditions, fibroblasts from sPD cells presented decreased basal respiration, ATP-linked OCR and maximal respiration, and increased mitochondria-targeting phosphorylation of DRP1 when compared to control cells. Our work validates the relevance of using fibroblasts from sPD patients to study cellular and molecular changes that are characteristic of dopaminergic neurodegeneration of PD, and shows that forcing mitochondrial OXPHOS uncovers metabolic defects that were otherwise hidden.

Early Cardiac Mitochondrial Molecular and Functional Responses to Acute Anthracycline Treatment in Wistar Rats.

Doxorubicin (DOX) is an anticancer drug widely used to treat human and nonhuman tumors but the late and persistent cardio-toxicity reduces the therapeutic utility of the drug. The full mechanism(s) of DOX-induced acute, subchronic and delayed toxicity, which has a preponderant mitochondrial component, remains unclear; therefore, it is clinically relevant to identify early markers to identify patients who are predisposed to DOX-related cardiovascular toxicity. To address this, Wistar rats (16 weeks old) were treated with a single DOX dose (20 mg/kg, i.p.); then, mRNA, protein levels and functional analysis of mitochondrial endpoints were assessed 24 h later in the heart, liver, and kidney. Using an exploratory data analysis, we observed cardiac-specific alterations after DOX treatment for mitochondrial complexes III, IV, and preferentially for complex I. Conversely, the same analysis revealed complex II alterations are associated with DOX response in the liver and kidney. Interestingly, H2O2 production by the mitochondrial respiratory chain as well as loss of calcium-loading capacity, markers of subchronic toxicity, were not reliable indicators of acute DOX cardiotoxicity in this animal model. By using sequential principal component analysis and feature correlation analysis, we demonstrated for the first time alterations in sets of transcripts and proteins, but not functional measurements, that might serve as potential early acute markers of cardiac-specific mitochondrial toxicity, contributing to explain the trajectory of DOX cardiac toxicity and to develop novel interventions to minimize DOX cardiac liabilities.

Multidimensional knapsack problem: a fitness landscape analysis.

Fitness landscape analysis techniques are used to better understand the influence of genetic representations and associated variation operators when solving a combinatorial optimization problem. Five representations are investigated for the multidimensional knapsack problem. Common mutation operators, such as bit-flip mutation, are employed to generate fitness landscapes. Measures such as fitness distance correlation and autocorrelation are applied to examine the landscapes associated with the tested genetic encodings. Furthermore, additional experiments are made to observe the effects of adding heuristics and local optimization to the representations. Encodings with a strong heuristic bias are more efficient, and the addition of local optimization techniques further enhances their performance.

Industrial robust yeast isolates with great potential for fermentation of lignocellulosic biomass.

The search of robust microorganisms is essential to design sustainable processes of second generation bioethanol. Yeast strains isolated from industrial environments are generally recognised to present an increased stress tolerance but no specific information is available on their tolerance towards inhibitors that come from the pretreatment of lignocellulosic materials. In this work, a strategy for the selection of different yeasts using hydrothermal hydrolysate from Eucalyptus globulus wood, containing different concentrations of inhibitors, was developed. Ten Saccharomyces cerevisiae and four Kluyveromyces marxianus strains isolated from industrial environments and four laboratory background strains were evaluated. Interestingly, a correlation between final ethanol titer and percentage of furfural detoxification was observed. The results presented here highlight industrial distillery environments as a remarkable source of efficient yeast strains for lignocellulosic fermentation processes. Selected strains were able to resourcefully degrade furfural and HMF inhibitors, producing 0.8g ethanol/Lh corresponding to 94% of the theoretical yield.

Plasmid-mediate transfer of FLO1 into industrial Saccharomyces cerevisiae PE-2 strain creates a strain useful for repeat-batch fermentations involving flocculation-sedimentation.

The flocculation gene FLO1 was transferred into the robust industrial strain Saccharomyces cerevisiae PE-2 by the lithium acetate method. The recombinant strain showed a fermentation performance similar to that of the parental strain. In 10 repeat-batch cultivations in VHG medium with 345 g glucose/L and cell recycling by flocculation-sedimentation, an average final ethanol concentration of 142 g/L and an ethanol productivity of 2.86 g/L/h were achieved. Due to the flocculent nature of the recombinant strain it is possible to reduce the ethanol production cost because of lower centrifugation and distillation costs.

Robust industrial Saccharomyces cerevisiae strains for very high gravity bio-ethanol fermentations.

The application and physiological background of two industrial Saccharomyces cerevisiae strains, isolated from harsh industrial environments, were studied in Very High Gravity (VHG) bio-ethanol fermentations. VHG laboratory fermentations, mimicking industrially relevant conditions, were performed with PE-2 and CA1185 industrial strains and the CEN.PK113-7D laboratory strain. The industrial isolates produced remarkable high ethanol titres (>19%, v/v) and accumulated an increased content of sterols (2 to 5-fold), glycogen (2 to 4-fold) and trehalose (1.1-fold), relatively to laboratory strain. For laboratory and industrial strains, a sharp decrease in the viability and trehalose concentration was observed above 90 g l⁻¹ and 140 g l⁻¹ ethanol, respectively. PE-2 and CA1185 industrial strains presented important physiological differences relatively to CEN.PK113-7D strain and showed to be more prepared to cope with VHG stresses. The identification of a critical ethanol concentration above which viability and trehalose concentration decrease significantly is of great importance to guide VHG process engineering strategies. This study contributes to the improvement of VHG processes by identifying yeast isolates and gathering yeast physiological information during the intensified fermentation process, which, besides elucidating important differences between these industrial and laboratory strains, can drive further process optimization.

Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations.

BACKGROUND: The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions. RESULTS: Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of Bud31 and Hpr1 was found to lead to the increase of both ethanol yield and fermentation rate, while Pho85, Vrp1 and Ygl024w expression is required for maximal ethanol production in VHG fermentations. Five genes, Erg2, Prs3, Rav1, Rpb4 and Vma8, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate. CONCLUSIONS: The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers.

Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs.

Statistical experimental designs were used to develop a medium based on corn steep liquor (CSL) and other low-cost nutrient sources for high-performance very high gravity (VHG) ethanol fermentations by Saccharomyces cerevisiae. The critical nutrients were initially selected according to a Plackett-Burman design and the optimized medium composition (44.3 g/L CSL; 2.3 g/L urea; 3.8 g/L MgSO4·7H2O; 0.03 g/L CuSO4·5H2O) for maximum ethanol production by the laboratory strain CEN.PK 113-7D was obtained by response surface methodology, based on a three-level four-factor Box-Behnken design. The optimization process resulted in significantly enhanced final ethanol titre, productivity and yeast viability in batch VHG fermentations (up to 330 g/L glucose) with CEN.PK113-7D and with industrial strain PE-2, which is used for bio-ethanol production in Brazil. Strain PE-2 was able to produce 18.6±0.5% (v/v) ethanol with a corresponding productivity of 2.4±0.1g/L/h. This study provides valuable insights into cost-effective nutritional supplementation of industrial fuel ethanol VHG fermentations.