How does the geometry affect the internal biomechanics of a lumbar spine bi-segment finite element model? Consequences on the validation process

Numerical modelling can provide a thorough understanding of the mechanical influence on the spinal tissues and may offer explanations to mechanically linked pathologies. Such objective might be achieved only if the models are carefully validated. Sensitivity study must be performed in order to evaluate the influence of the approximations inherent to modelling. In this study, a new geometrically accurate L3-L5 lumbar spine bi-segmental finite-element model was acquired by modifying a previously existing model. The effect of changes in bone geometry, ligament fibres distribution, nucleus position and disc height was investigated in flexion and extension by comparison of the results obtained from the model before and after the geometrical update. Additional calculations were performed in axial rotation and lateral bending in order to compare the computed ranges of motion (ROM) with experimental results. It was found that the geometrical parameters affected the stress distribution and strain energy in the zygapophysial joints, the ligaments, and the intervertebral disc, changing qualitatively and quantitatively their relative role in resisting the imposed loads. The predicted ROM were generally in good agreement with the experimental results, independently of the geometrical changes. Hence, although the model update affected its internal biomechanics, no conclusions could be drawn from the experimental data about the validation of a particular geometry. Hence the validation of the lumbar spine model should be based on the relative role of its structural components and not only on its global mobility.     

CYP27A1 and CYP24 expression as a function of malignant transformation in the colon.

Vitamin D deficiency is strongly associated with the risk of developing colorectal cancer (CRC). Because of the propensity of bioactive 1,25-dihydroxyvitamin D3 to cause toxic hypercalcemia, considerable effort has been directed to identifying safer drugs while retaining the efficacy of the parent compound. However, vitamin D precursors do not present toxicity concerns and may be sufficient for CRC chemoprevention or chemotherapy, providing the appropriate enzymes are present in colonic epithelia. We previously showed that CYP27B1 is present at equally high levels in the colon and CRC irrespective of differentiation but was not present in metastases. In this study we used quantitative immunohistochemistry to show that CYP27A1, converting D3 to 25-hydroxycholecalciferol, is present in increasing concentrations in the nuclei of normal colonic epithelia, aberrant crypt foci (ACF), and adenomatous polyps. Whereas total cellular CYP27A1 remains high in CRC and lymph node metastases, the amount of enzyme present in the nuclei decreases with tumor cell dedifferentiation while rising in the cytoplasm. Similarly, increasing amounts of the deactivating enzyme CYP24 are present in the nuclei of normal colonic epithelia, ACFs, and adenomatous polyps. Although the amount of total CYP24 decreases slightly in CRC as a function of tumor cell dedifferentiation and metastasis, location of this enzyme shifts almost entirely from the nuclear compartment to the cytoplasmic compartment. These data indicate that non-toxic vitamin D precursors should be sufficient for CRC chemoprevention, but that neither vitamin D nor its precursors may be sufficient for CRC chemotherapy.     

AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

The initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.     

The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.     

Transition metal complexes bearing atropisomeric saturated NHC ligands

From achiral imidazolinium salts, chiral transition metal complexes containing an N-heterocyclic carbene (NHC) ligand were prepared (metal = palladium, copper, silver, gold, rhodium). Axial chirality in these complexes results from the formation of the metal-carbene bond leading to the restriction of rotation of dissymmetric N-aryl substituents about the C–N bond. When these complexes exhibited a sufficient configurational stability, a resolution by chiral high-performance liquid chromatography (HPLC) on preparative scale enabled isolation of enantiomers with excellent enantiopurities (>99% ee) and good yields. A study of the enantiomerization barriers revealed the effect of the backbone nature as well as the type of transition metal on its values. Nevertheless, the evaluation of palladium-based complexes in asymmetric intramolecular α-arylation of amides demonstrated that the ability to induce an enantioselectivity cannot be correlated to the configurational stability of the precatalysts.     

Racemization mechanism of lithium tert-butylphenylphosphido-borane: A kinetic insight

The racemization mechanism of tert-butylphenylphosphido-borane is investigated experimentally and theoretically. Based on this converging approach, it is shown, first, that several phosphido-borane molecular species coexist at the time of the reaction and, second, that one particular of both initially assumed reactive routes most significantly contribute to the overall racemization process. From our converging modeling and experimental measurement, it comes out that the most probable species to be here encountered is a phosphido-borane-Li (THF)₂ neutral solvate, whose P-stereogenic center monomolecular inversion through a Y-shaped transition structure (Δ_rG°^≠: 81 kJ mol^?1) brings the largest contribution to the racemization process.     

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP⁺ oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.Unicellular photosynthetic organisms such as the green alga Chlamydomonas reinhardtii frequently experience anoxic conditions in their natural habitat, especially during the night when the microbial community consumes the available oxygen. Under anoxia, lack of ATP synthesis by F₁F_O ATP synthase (EC 3.6.3.14) due to the absence of mitochondrial respiration is compensated by the activity of various plant- and bacterial-type fermentative enzymes that drive a sustained glycolytic activity (Mus et al., 2007; Terashima et al., 2010; Grossman et al., 2011; Yang et al., 2014). In C. reinhardtii, upstream glycolytic enzymes, including the reversible glyceraldehyde 3-P dehydrogenase, are located in the chloroplast (Johnson and Alric, 2012). This last enzyme is shared by the glycolysis (oxidative activity) and the Calvin-Benson-Bassham (CBB) cycle (reductive activity; Johnson and Alric, 2013). In dark anoxic conditions, the CBB cycle is inactive, thus avoiding wasteful using up of available ATP and depletion of the required intermediates for glycolysis. On the other side, ability of microalgae to perform photosynthetic carbon fixation when transferred from dark to light in the absence of oxygen might also be critical for adaptation to their environment. In such conditions, not only the linear electron flow (LEF) to Rubisco, but also alternative electron flow (AEF) toward oxygen (chlororespiration, Mehler reaction, and mitochondrial respiration; for review, see Miyake, 2010; Peltier et al., 2010; Cardol et al., 2011) is impaired. Thus, cells need to circumvent a paradoxical situation: the activity of the CBB cycle requires the restoration of the cellular ATP, but the chloroplastic F₁F_O ATP synthase activity is compromised by the impairment of most of the photosynthetic electron flows that usually generate the proton motive force in oxic conditions. Other AEFs, specific to anoxic conditions, should therefore be involved to promote ATP synthesis without net synthesis of NADPH and explain the light-induced restoration of CBB cycle activity.Among enzymes expressed in anoxia, the oxygen-sensitive hydrogenases (HYDA1 and HYDA2 in C. reinhardtii) catalyze the reversible reduction of protons into molecular hydrogen from the oxidation of reduced ferredoxins (FDXs; Florin et al., 2001). Although hydrogen metabolism in microalgae has been largely studied in the last 15 years in perspective of promising future renewable energy carriers (Melis et al., 2000; Kruse et al., 2005; Ghirardi et al., 2009), the physiological role of such an oxygen-sensitive enzyme linked to the photosynthetic pathway has been poorly considered. The 40-year-old proposal that H₂ evolution by hydrogenase is involved in induction of photosynthetic electron transfer after anoxic incubation (Kessler, 1973; Schreiber and Vidaver, 1974) has been only recently demonstrated in C. reinhardtii. Gas exchange measurements showed that H₂ evolution occurs prior to CO₂ fixation upon illumination (Cournac et al., 2002). At light onset after a prolonged period in dark anoxic conditions, the photosynthetic electron flow is mainly a LEF toward hydrogenase (Godaux et al., 2013), and lack of hydrogenase activity in hydrogenase maturation factor EF (hydEF) mutant strain deficient in hydrogenases maturation (Posewitz et al., 2004) induces a lag in induction of PSII activity (Ghysels et al., 2013). In cyanobacteria, the bidirectional Ni-Fe hydrogenase might also work as an electron valve for disposal of electrons generated at the onset of illumination of cells (Cournac et al., 2004) or when excess electrons are generated during photosynthesis, preventing the slowing of the electron transport chain under stress conditions (Appel et al., 2000; Carrieri et al., 2011). The bidirectional Ni-Fe hydrogenase could also dispose of excess of reducing equivalents during fermentation in dark anaerobic conditions, helping to generate ATP and maintaining homeostasis (Barz et al., 2010). A similar role for hydrogenase in setting the redox poise in the chloroplast of C. reinhardtii in anoxia has been recently uncovered (Clowez et al., 2015).Still, the physiological and evolutionary advantages of hydrogenase activity have not been demonstrated so far, and the mechanism responsible for the cessation of hydrogen evolution remains unclear. In this respect, at least three hypotheses have been formulated: (1) the inhibition of hydrogenase by O₂ produced by water photolysis (Ghirardi et al., 1997; Cohen et al., 2005), (2) the competition between ferredoxin-NADP⁺ oxidoreductase (FNR) and hydrogenase activity for reduced FDX (Yacoby et al., 2011), and (3) the inhibition of electron supply to hydrogenases by the proton gradient generated by another AEF, the cyclic electron flow around PSI (PSI-CEF; Tolleter et al., 2011). First described by Arnon (1955), PSI-CEF consists in a reinjection of electrons from reduced FDX or NADPH pool in the plastoquinone (PQ) pool. By generating an additional transthylakoidal proton gradient without producing reducing power, this AEF thus contributes to adjust the ATP/NADPH ratio for carbon fixation in various energetic unfavorable conditions including anoxia (Tolleter et al., 2011; Alric, 2014), high light (Tolleter et al., 2011; Johnson et al., 2014), or low CO₂ (Lucker and Kramer, 2013). In C. reinhardtii, two pathways have been suggested to be involved in PSI-CEF: (1) a type II NAD(P)H dehydrogenase (NDA2; Jans et al., 2008) driving the electrons from NAD(P)H to the PQ pool and (2) a pathway involving Proton Gradient Regulation (PGR) proteins where electrons from reduced FDXs return to the PQ pool or cytochrome b₆f. Not fully understood, this latter pathway comprises at least Proton Gradient Regulation5 (PGR5) and Proton-Gradient Regulation-Like1 (PGRL1) proteins (Iwai et al., 2010; Tolleter et al., 2011; Johnson et al., 2014) and is the major route for PSI-CEF in C. reinhardtii cells placed in anoxia (Alric, 2014).In this work, we took advantage of specific C. reinhardtii mutants defective in hydrogenase activity and PSI-CEF to study photosynthetic electron transfer after a period of dark anoxic conditions. Based on biophysical and physiological complementary studies, we demonstrate that at least hydrogenase activity or PSI-CEF is compulsory for the activity of the CBB cycle and for the survival of the cells submitted to anoxic conditions in their natural habitat.     

Mono- and Dialkyl Glycerol Ether Lipids in Anaerobic Bacteria: Biosynthetic Insights from the Mesophilic Sulfate Reducer Desulfatibacillum alkenivorans PF2803T

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803^T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1′-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803^T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1′-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.