A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose.

High-temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the ß-glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non-axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline-treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by-products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose-derived sugars by C. vulgaris under mixotrophic growth.

Growth Temperature Influence on Lipids and Photosynthesis in Lepidium sativum.

Temperature has a major impact on plant development and growth. In temperate climates, the seasonal temperature displays large variations that can affect the early stages of plant growth and development. Sessile organisms need to be capable of responding to these conditions, so that growth temperature induces morphological and physiological changes in the plant. Besides development, there are also important molecular and ultrastructural modifications allowing to cope with different temperatures. The chloroplast plays a crucial role in plant energetic metabolism and harbors the photosynthetic apparatus. The photosynthetic light reactions are at the interface between external physical conditions (light, temperature) and the cell biochemistry. Therefore, photosynthesis requires structural flexibility to be able to optimize its efficiency according to the changes of the external conditions. To investigate the effect of growth temperature on the photosynthetic apparatus, we followed the photosynthetic performances and analyzed the protein and lipid profiles of Lepidium sativum (cress) grown at three different temperatures. This revealed that plants developing at temperatures above the optimum have a lower photosynthetic efficiency. Moreover, plants grown under elevated and low temperatures showed a different galactolipid profile, especially the amount of saturated galactolipids decreased at low temperature and increased at high temperature. From the analysis of the chlorophyll a fluorescence induction, we assessed the impact of growth temperature on the re-oxidation of plastoquinone, which is the lipidic electron carrier of the photosynthetic electron transport chain. We show that, at low temperature, along with an increase of unsaturated structural lipids and plastochromanol, there is an increase of the plastoquinone oxidation rate in the dark. These results emphasize the importance of the thylakoid membrane composition in preserving the photosynthetic apparatus under non-optimal temperatures.

Regulation of Light Harvesting in Chlamydomonas reinhardtii Two Protein Phosphatases Are Involved in State Transitions.

Protein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer, and during state transitions, the kinase STATE TRANSITION7 (STT7) of Chlamydomonas reinhardtii phosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to PSI or PSII, depending on light conditions and metabolic demands, but counteracting phosphatase(s) remain unknown in C. reinhardtii Here we analyzed state transitions in C. reinhardtii mutants of two phosphatases, PROTEIN PHOSPHATASE1 and PHOTOSYSTEM II PHOSPHATASE, which are homologous to proteins that antagonize the state transition kinases (STN7 and STN8) in Arabidopsis (Arabidopsis thaliana). The transition from state 2 to state 1 was retarded in pph1, and surprisingly also in pbcp However, both mutants eventually returned to state 1. In contrast, the double mutant pph1;pbcp appeared strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits were affected in the phosphatase mutants. Their analysis indicated that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein dephosphorylation and the more complex antenna phosphorylation patterns in C. reinhardtii compared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga.

Mutation of the Atypical Kinase ABC1K3 Partially Rescues the PROTON GRADIENT REGULATION 6 Phenotype in Arabidopsis thaliana.

Photosynthesis is an essential pathway providing the chemical energy and reducing equivalents that sustain higher plant metabolism. It relies on sunlight, which is an inconstant source of energy that fluctuates in both intensity and spectrum. The fine and rapid tuning of the photosynthetic apparatus is essential to cope with changing light conditions and increase plant fitness. Recently PROTON GRADIENT REGULATION 6 (PGR6-ABC1K1), an atypical plastoglobule-associated kinase, was shown to regulate a new mechanism of light response by controlling the homeostasis of photoactive plastoquinone (PQ). PQ is a crucial electron carrier existing as a free neutral lipid in the photosynthetic thylakoid membrane. Perturbed homeostasis of PQ impairs photosynthesis and plant acclimation to high light. Here we show that a homologous kinase, ABC1K3, which like PGR6-ABC1K1 is associated with plastoglobules, also contributes to the homeostasis of the photoactive PQ pool. Contrary to PGR6-ABC1K1, ABC1K3 disfavors PQ availability for photosynthetic electron transport. In fact, in the abc1k1/abc1k3 double mutant the pgr6(abc1k1) the photosynthetic defect seen in the abc1k1 mutant is mitigated. However, the PQ concentration in the photoactive pool of the double mutant is comparable to that of abc1k1 mutant. An increase of the PQ mobility, inferred from the kinetics of its oxidation in dark, contributes to the mitigation of the pgr6(abc1k1) photosynthetic defect. Our results also demonstrate that ABC1K3 contributes to the regulation of other mechanisms involved in the adaptation of the photosynthetic apparatus to changes in light quality and intensity such as the induction of thermal dissipation and state transitions. Overall, we suggests that, besides the absolute concentration of PQ, its mobility and exchange between storage and active pools are critical for light acclimation in plants.

The Kinase STATE TRANSITION 8 Phosphorylates Light Harvesting Complex II and Contributes to Light Acclimation in Arabidopsis thaliana.

Phosphorylation of the light-harvesting complex II (LHCII) is a central trigger for the reorganization of the photosynthetic complexes in the thylakoid membrane during short-term light acclimation. The major kinase involved in LHCII phosphorylation is STATE TRANSITION 7 (STN7), and its activity is mostly counteracted by a thylakoid-associated phosphatase, PROTEIN PHOSPHATASE 1/THYLAKOID ASSOCIATED PHOSPHATASE 38 (PPH1/TAP38). This kinase/phosphatase pair responds to the redox status of the photosynthetic electron transport chain. In Arabidopsis thaliana, Lhcb1 and Lhcb2 subunits of the LHCII trimers are the major targets of phosphorylation and have different roles in the acclimation of the photosynthetic machinery. Another antagonistic kinase and phosphatase pair, STATE TRANSITION 8 (STN8) and PHOTOSYSTEM II PHOSPHATASE (PBCP) target a different set of thylakoid proteins. Here, we analyzed double, triple, and quadruple knockout mutants of these kinases and phosphatases. In multiple mutants, lacking STN7, in combination with one or both phosphatases, but not STN8, the phosphorylation of LHCII was partially restored. The recovered phosphorylation favors Lhcb1 over Lhcb2 and results in a better adaptation of the photosynthetic apparatus and increased plant growth under fluctuating light. This set of mutants allowed to unveil a contribution of STN8-dependent phosphorylation in the acclimation to rapid light variations.

How chloroplasts protect themselves from unfolded proteins.

A genetic screen has identified the first signaling component of the unfolded protein response in chloroplasts.

Plastoquinone homoeostasis by Arabidopsis proton gradient regulation 6 is essential for photosynthetic efficiency.

Photosynthesis produces organic carbon via a light-driven electron flow from H2O to CO2 that passes through a pool of plastoquinone molecules. These molecules are either present in the photosynthetic thylakoid membranes, participating in photochemistry (photoactive pool), or stored (non-photoactive pool) in thylakoid-attached lipid droplets, the plastoglobules. The photoactive pool acts also as a signal of photosynthetic activity allowing the adaptation to changes in light condition. Here we show that, in Arabidopsis thaliana, proton gradient regulation 6 (PGR6), a predicted atypical kinase located at plastoglobules, is required for plastoquinone homoeostasis, i.e. to maintain the photoactive plastoquinone pool. In a pgr6 mutant, the photoactive pool is depleted and becomes limiting under high light, affecting short-term acclimation and photosynthetic efficiency. In the long term, pgr6 seedlings fail to adapt to high light and develop a conditional variegated leaf phenotype. Therefore, PGR6 activity, by regulating plastoquinone homoeostasis, is required to cope with high light.

Comparison of transplastomic Chlamydomonas reinhardtii and Nicotiana tabacum expression system for the production of a bacterial endoglucanase.

The bulk production of recombinant enzymes by either prokaryotic or eukaryotic organisms might contribute to replace environmentally non-friendly chemistry-based industrial processes with enzyme-based biocatalysis, provided the cost of enzyme production is low. In this context, it is worth noting that the production of recombinant proteins by photosynthetic organisms offer both eukaryotic (nuclear) and prokaryotic (chloroplast) alternatives, along with the advantage of an autotrophic nutrition. Compared to nuclear transformation, chloroplast transformation generally allows a higher level of accumulation of the recombinant protein of interest. Furthermore, among the photosynthetic organisms, there is a choice of using either multicellular or unicellular ones. Tobacco, being a non-food and non-feed plant, has been considered as a good choice for producing enzymes with applications in technical industry, using a transplastomic approach. Also, unicellular green algae, in particular Chlamydomonas reinhardtii, have been proposed as candidate organisms for the production of recombinant proteins. In the light of the different features of these two transplastomic systems, we decided to make a direct comparison of the efficiency of production of a bacterial endoglucanase. With respect to the amount obtained, 14 mg g-1 of biomass fresh weight equivalent to 8-10% of the total protein content and estimated production cost, 1.5-2€ kg-1, tobacco proved to be far more favorable for bulk enzyme production when compared to C. reinhardtii which accumulated this endoglucanase at 0.003% of the total protein.

Use of Nicotiana tabacum transplastomic plants engineered to express a His-tagged CP47 for the isolation of functional photosystem II core complexes.

This work focuses on the development of a molecular tool for purification of Photosystem II (PSII) from Nicotiana tabacum (L.). To this end, the chloroplast psbB gene encoding the CP47 PSII subunit was replaced with an engineered version of the same gene containing a C-terminal His-tag. Molecular analyses assessed the effective integration of the recombinant gene and its expression. Despite not exhibiting any obvious phenotype, the transplastomic plants remained heteroplasmic even after three rounds of regeneration under antibiotic selection. However, the recombinant His-tagged CP47 protein associated in vivo to the other PSII subunits allowing the isolation of a functional PSII core complex, although with low yield of extraction. These results will open up possible perspectives for further spectroscopic and structural studies.

A Nucleus-Encoded Chloroplast Phosphoprotein Governs Expression of the Photosystem I Subunit PsaC in Chlamydomonas reinhardtii.

The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half-A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5' untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5'end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain.

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex.

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b6f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b6f complex.

Improving cardiovascular prevention in general practice: Results of a comprehensive personalized strategy in subjects at high risk.

BACKGROUND: Although high cardiovascular risk patients should be the main target of preventive strategies, modifiable risk factors are often inadequately controlled. AIM: To assess feasibility and results of a comprehensive personalized method for cardiovascular prevention in high risk patients followed by their general practitioner. METHODS: Between 2004 and 2007, 12,513 patients (mean age 64.0 ± 9.5 years; 61.5% males) with multiple cardiovascular risk factors or history of atherosclerotic disease were identified and followed for five years. If control of major modifiable cardiovascular risk factors (hypertension, hypercholesterolaemia, diabetes, obesity, smoking, unhealthy diet, physical inactivity) was sub-optimal, at baseline and yearly thereafter general practitioners planned with patients, with the help of a brief checklist, preventive interventions to improve the global risk profile. Main outcome was the control of the seven major modifiable cardiovascular risk factors during follow-up. Secondary outcome was the incidence of cardiovascular deaths and hospitalization for cardiovascular reasons according to the improvement in global cardiovascular risk profile during the first year. RESULTS: Control of all major modifiable risk factors except physical inactivity improved gradually and significantly (p < 0.0001) during follow-up.The improvement in the global cardiovascular risk profile during the first year was independently and significantly associated with a lower rate of major cardiovascular events in the following years (hazard ratio 0.939; 95% confidence interval 0.887-0.994, p = 0.03). CONCLUSIONS: Our comprehensive, personalized method for cardiovascular risk prevention in people at high risk appears feasible in general practice. The improvement in the global cardiovascular risk profile was associated with a better prognosis.

Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions.

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.

Production by Tobacco Transplastomic Plants of Recombinant Fungal and Bacterial Cell-Wall Degrading Enzymes to Be Used for Cellulosic Biomass Saccharification.

Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2 increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

Graft monocytic myeloid-derived suppressor cell content predicts the risk of acute graft-versus-host disease after allogeneic transplantation of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells.

Myeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Lin(low/neg)HLA-DR(-)CD11b(+)CD33(+)CD14(+)) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD.

Light-dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes.

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light-harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non-photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1-2. This protein, which showed light intensity-dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light-induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI-PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.

Peripheral blood CD34+ cell monitoring after cyclophosphamide and granulocyte-colony-stimulating factor: an algorithm for the pre-emptive use of plerixafor.

Plerixafor "on demand" after chemotherapy plus granulocyte-colony-stimulating factor (G-CSF) is efficient in peripheral stem cell mobilization, but the timing of administration and criteria for patient selection are under investigation. To devise an algorithm for the "on demand" use of plerixafor at the first mobilization attempt, we analyzed the kinetics of hematopoietic recovery and peripheral blood CD34+ cells in 107 patients treated with high-dose cyclophosphamide plus G-CSF. Fifty-one patients with myeloma were treated with cyclophosphamide 3-4 g/m(2) on day 0 followed by G-CSF 10 µg/kg from day + 6, and 56 patients with lymphoma received cyclophosphamide 6-7 g/m(2) followed by G-CSF 5 µg/kg from day + 1. Peripheral blood CD34+ cell monitoring was started on day + 8 in patients with myeloma and day + 10 in patients with lymphoma. The outcome of interest was a collection of ≤ 2 × 10(6) CD34+/kg. By a multivariate logistic regression model, CD34+ cell count < 10/µL at leukocyte recovery (> 1000/µL) or leukocyte count < 1000/µL after day + 12 in myeloma and day + 14 in lymphoma predicted the failure of mobilization by 2.7 and 2.8 times (p = 0.001 and p = 0.02) with a sensitivity of 89% and specificity of 88%, respectively. Plerixafor "on demand" may be considered in patients with myeloma and lymphoma with delayed hematopoietic recovery and < 10/µL CD34+ cells, as a first-line mobilization strategy.

Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme.

The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC 3.2.1.8), a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage.

Growth and lipid synthesis promotion in mixotrophic Neochloris oleoabundans (Chlorophyta) cultivated with glucose.

In the recent years, the studies concerning the cultivation of Neochloris oleoabundans for biofuel purposes have increased, in relation to its capability to accumulate lipids when grown under nutrient starvation. Unfortunately, this cultivation mode does not allow to reach high biomass densities, which are required to improve the feasibility of the process. Increasing knowledge of the microalgal physiology is necessary to obtain new useful information for the improvement of culture performance in the perspective of large-scale cultivation. In this work, the mixotrophic cultivation of N. oleoabundans in a brackish medium added with different glucose concentrations has been tested under shaking, with the aim of stimulating growth alongside lipid accumulation inside cells. Cell morphology, glucose consumption, photosynthetic pigment content and photosynthetic efficiency were also investigated. Among all tested glucose concentrations (0-30 g L(-1)), it was observed that 2.5 g L(-1) was the optimal concentration, allowing to obtain the best compromise between glucose supplement, biomass production and lipid accumulation. Growth was highly enhanced in mixotrophic cultures, linked to the release of cells from sporocysts. A unique feature characterising mixotrophy in N. oleoabundans was the promotion of the maximum quantum yield of Photosystem II. Moreover, when mixotrophic cells entered the stationary phase, high lipid accumulation was induced. This study shows that the addition of glucose to N. oleoabundans remarkably increases the production of biomass enriched in lipids and represents an advancement for the cultivation of this microalga for applied purposes.

Predictors of CD34+ cell mobilization and collection in adult men with germ cell tumors: implications for the salvage treatment strategy.

BACKGROUND: High-dose chemotherapy with tandem or triple carboplatin and etoposide course is currently the first curative choice for relapsing GCT. The collection of an adequate amount of hematopoietic (CD34(+)) stem cells is a priority. PATIENTS AND METHODS: We analyzed data of patients who underwent HDCT at 2 referral institutions. Chemotherapy followed by myeloid growth factors was applied in all cases. Uni- and multivariable models were used to evaluate the association between 2 prespecified variables and mobilization parameters. Analyses included only the first mobilizing course of chemotherapy and mobilization failures. RESULTS: A total of 116 consecutive patients underwent a mobilization attempt from December 1995 to November 2012. Mobilizing regimens included cyclophosphamide (CTX) 7 gr/m(2) (n = 39), cisplatin, etoposide, and ifosfamide (PEI) (n = 42), paclitaxel, cisplatin, and gemcitabine (TPG) (n = 11), and mixed regimens (n = 24). Thirty-seven percent were treated in first-line, 50% (n = 58) in second-line, 9.5% (n = 11) and 3.4% (n = 4) in third- and fourth-line settings, respectively. Six patients did not undergo HDCT because they were poor mobilizers, 2 in first- and second-line (1.9%), and 4 beyond the second-line (26.7%). In the multivariable model, third-line or later setting was associated with a lower CD34(+) cell peak/µL (P = .028) and a lower total CD34(+)/kg collected (P = .008). The latter was also influenced by the type of mobilizing regimen (P < .001). CONCLUSION: A decline in significant mobilization parameters was found, primarily depending on the pretreatment load. Results lend support to the role of CD34(+) cell mobilization in the therapeutic algorithm of relapsing GCT, for whom multiple HDCT courses are still an option, and potentially a cure.