The role of plasma membrane STIM1 and Ca2 +entry in platelet aggregation. STIM1 binds to novel proteins in human platelets

Ca^2 + elevation is essential to platelet activation. STIM1 senses Ca^2 + in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca^2 + entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca^2 + entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca^2 + entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca^2 +-sensing role of STIM1 is served by the protein in the ER.     

The influence of light quality on akinete formation and germination in the toxic cyanobacterium Anabaena circinalis

Physiological control of akinete formation and subsequent germination is likely to be important in understanding and predicting how natural populations of cyanobacteria respond to their environment. While previous research has indicated nutrient limitation may be important in akinete formation new results presented here indicate that in the toxic and bloom-forming species Anabaena circinalis there was a profound effect of spectral quality. Under 40 μmol photons m^?2 s^?1 photosynthetically active irradiance (PAR) of predominately red irradiance akinete production was maximal at 2.1 × 10^?4 akinetes vegetative cell^?1 d^?1, some 3000 times greater than the 6.5 × 10^?8 akinetes vegetative cell^?1 d^?1 observed under equivalent PAR but predominately blue light. For cells grown under a range of predominantly red, white and green irradiance even short exposures to blue light reduced akinete formation rates by a factor of ten relative to controls, indicating that exposure to blue light inhibits akinete formation. Germination of akinetes was not influenced by the irradiance under which akinetes were formed: 88 ± 4.1% (mean ± 1 S.D.) of akinetes germinated with no evidence of an effect on germination success due to their production under predominately red, white or green irradiance (germination of akinetes produced under blue light was not tested). Spectral quality had a significant impact on both vegetative cell and germling growth rates. The results indicate a significant reduction in the cellular differentiation of A. circinalis vegetative cells into akinetes that is mediated by blue light. In an ecological context the production of akinetes will be greater in environments with less blue light; potentially including those with slower flow, more stratification, less vertical mixing and more turbidity. The resulting spatial pattern of akinete production is likely to influence the location of akinetes in sediments and the development of subsequent blooms from excysting germlings.     

Evaluation of mobilized peripheral stem cells according to CD34 and aldehyde dehydrogenase expression and effect of SSClo ALDHbr cells on hematopoietic recovery

Background aimsWe evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte–colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m² once daily, intravenously on day 1). We also investigated the relationship between the number of SSC^lo CD45^dim CD34^hi cells, SSC^lo ALDH^br cells and engraftment.MethodsThirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n% = 14), Hodgkin's lymphoma (n% = 7), non-Hodgkin's lymphoma (n% = 2), acute myloid leukemia (n% = 2), chronic lymphocytic leukemia (n% = 1) and germ cell testis tumor (n% = 1).ResultsNumbers of SSC^lo CD45^dim CD34^hi cells and SSC^lo ALDH^br cells were highly correlated in both peripheral blood and apheresis products (P < 0.001). We could not find a relationship between the transplanted SSC^lo CD45^dim CD34^hi cell dose or SSC^lo ALDH^br cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC^lo CD45^dim CD34^hi cells were 5.40 × 10⁶/kg for neutrophil recovery and 7.22 × 10⁶/kg for platelet recovery. The optimal thresholds for SSC^lo ALDH^br cells were 6.53 × 10⁶/kg for neutrophil recovery and 8.72 × 10⁶/kg platelet recovery.ConclusionsAccording to our data, numbers of SSC^lo ALDH^br cells are in very good agreement with numbers of SSC^lo CD45^dim CD34^hi cells and can be a predictor of stem cell mobilization.     

Proteomics analysis of proteins in green alga Haematococcus lacustris (Chlorophyceae) expressed under combined stress of nitrogen starvation and high irradiance

This study is aimed at identifying the proteins that are up-regulated during astaxanthin accumulation in Haematococcus lacustris. For this H. lacustris cells were cultivated in photobioreactors under normal light irradiance of 40 μE m^?2 s^?1 for 6 days and then induced to accumulate astaxanthin for 3 days further by exposure to continuous high irradiance of 200 μE m^?2 s^?1 with fluorescent lamps as light source after the cells reached the stationary phase in a nitrogen-depleted condition. Under this condition, the average astaxanthin content per cell increased from 91 mg/l up to 406 mg/l after 3 days of induction. The proteomics data from a two-dimensional electrophoretic comparison demonstrated that a combination of nitrogen source depletion and 1 h high light have significantly changed the pattern of protein expression in H. lacustris. A total of 49 protein spots were picked after 1 h of stress induction. They consisted of 13 down-regulated proteins and 36 up-regulated proteins. Fifteen proteins which had highly up-regulated expression were further analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results will point toward interesting proteins that can be pursued for further analysis of astaxanthin biosynthesis pathway.     

Influence of erythrocyte aggregation on radial migration of platelet-sized spherical particles in shear flow

Blood platelets when activated are involved in the mechanisms of hemostasis and thrombosis, and their migration toward injured vascular endothelium necessitates interaction with red blood cells (RBCs). Rheology co-factors such as a high hematocrit and a high shear rate are known to promote platelet mass transport toward the vessel wall. Hemodynamic conditions promoting RBC aggregation may also favor platelet migration, particularly in the venous system at low shear rates. The aim of this study was to confirm experimentally the impact of RBC aggregation on platelet-sized micro particle migration in a Couette flow apparatus. Biotin coated micro particles were mixed with saline or blood with different aggregation tendencies, at two shear rates of 2 and 10 s⁻¹ and three hematocrits ranging from 20 to 60%. Streptavidin membranes were respectively positioned on the Couette static and rotating cylinders upon which the number of adhered fluorescent particles was quantified. The platelet-sized particle adhesion on both walls was progressively enhanced by increasing the hematocrit (p < 0.001), reducing the shear rate (p < 0.001), and rising the aggregation of RBCs (p < 0.001). Particle count was minimum on the stationary cylinder when suspended in saline at 2 s⁻¹ (57 ± 33), and maximum on the rotating cylinder at 60% hematocrit, 2 s⁻¹ and the maximum dextran-induced RBC aggregation (2840 ± 152). This fundamental study is confirming recent hypotheses on the role of RBC aggregation on venous thrombosis, and may guide molecular imaging protocols requiring injecting active labeled micro particles in the venous flow system to probe human diseases.     

Parietin in the tolerant lichen Xanthoria parietina (L.) Th. Fr. increases protection of Trebouxia photobionts from cadmium excess

Secondary metabolites of lichens can be involved in production of chelates with heavy metals. We hypothesized that parietin plays important role in protection of photobiont cells in Xanthoria parietina from an excess of cadmium ions. Two types of X. parietina lichen thalli, natural with presence of secondary metabolite parietin (p+) as well as without parietin (p−) were exposed to different doses of cadmium (up to 300 μmol g⁻¹ dw). Based on determination of the total and intracellular Cd-accumulation, ergosterol and thiobarbituric acid reactive substances (TBARS) content did not show statistically significant differences in the response of both types of thalli (p+ and p−). However, a stronger toxic effect of the highest Cd-dose on photosynthetic pigment content and chlorophyll a fluorescence was observed in the parietin-depleted thalli. The protective role of parietin against Cd excess was better supported and concluded from the differences observed in the production of non-protein thiol compounds (cysteine, glutathione and phytochelatins) involved in Cd detoxification. In the p+ thalli Cys content was stable but GSH content slightly decreased in the studied Cd range, while in the p− thalli these compounds were completely absent at high Cd doses. At Cd doses higher than 37.5 μmol Cd g⁻¹ dw, toxic to both types of X. parietina thalli, Cys and GSH contents were significantly higher in p+ than in p− thalli. Also, the photobiont partner in the p+ thalli was better protected of the metal exposition, and able to produce phytochelatins (PCs) over the whole range of metal, while in the p− thalli the production was completely inhibited at 75 μmol Cd g⁻¹ dw and higher concentrations, together with the inhibition of cysteine (Cys) and reduced glutathione (GSH) production. The obtained results indicate that the parietin layer is a natural barrier decreasing Cd access to algal cells in X. parietina. Comparison of PCs production appeared to be the most sensitive marker for estimation of Cd availability to photobiont in the symbiotic system.     

Characterization of a recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in E. coli for enzyme replacement therapy of Morquio A disease

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Currently, specific therapies are not available for MPS IVA patients. In this study, a biologically active recombinant GALNS enzyme (rGALNS) produced in Escherichia coli was purified through a two-step chromatography process. The effect of temperature and pH on purified rGALNS stability was evaluated, as well as the stability in human serum. Finally, the uptake of rGALNS by HEK 293 cells and MPS IVA fibroblasts was evaluated. The use of a semi-continuous process allowed the production of an active extracellular rGALNS, which was used for protein purification. The purified rGALNS showed a specific activity of 0.29 U mg^?1 and a production yield of 0.78 mg L^?1. The rGALNS presented an optimal pH of 5.5 and was stable for 8 days at 4 °C. In human serum it was stable for up to 6 h. rGALNS was not taken up by the cultured cells, suggesting that N-linked oligosaccharides are not necessary for the production of an active enzyme or enzyme stability but for the cell uptake of protein. This study shows the first characterization of rGALNS produced by E. coli, and provides important information about purification, stability, and glycosylations effect for this type of enzymes.     

Potential use of nylon scouring pad cubes attachment method for pectinase production by Aspergillus niger HFD5A-1

This study investigated the novel use of scouring pad cubes as a support matrix for immobilization of fungal cell to enhance the pectinase production. Nylon scouring pad cubes were used for immobilized Aspergillus niger HFD5A-1 cells for pectinase production in flask submerge fermentation system. The enzyme activity of immobilized cell in scouring pad cubes gave higher activity compared to free cells. Various physical parameters for culture condition were studied to evaluate its effects on pectinase production. The maximum enzyme activity obtained was 11.05 U/mL on the 6th day of cultivation after using the optimized parameters of 6 scouring pad cubes, 1 × 10⁷ spores/mL of inoculum size, agitation speed of 150 rpm and incubated at 30 °C. The use of nylon scouring pad cubes gave an increment of about 335.0% of pectinase production (11.05 U/mL) compared to free cells (2.54 U/mL). The results therefore show scouring pad cubes could be a favorable carrier to immobilize the fungal cells for higher enzyme production in submerged fermentation.     

The simultaneous utilization of kinetic analysis and flow cytometry in the assessment of Lactobacillus rhamnosus ATCC 7469 physiological states produced by increasing oxygen limitation levels and lactic acid accumulation

Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h⁻¹ and 0.6 h⁻¹. At 0.45 h⁻¹, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD⁺ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D = 0.20 h⁻¹, 8% at D = 0.30 h⁻¹, 14% at D = 0.45 h⁻¹ and 26% for D = 0.62 h⁻¹, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.     

Sulfate reduction and microbial community of autotrophic biocathode in response to acidity

Microbial electrolysis cells (MECs) with autotrophic biocathode are a promising technology for removal of pollutants in wastewater. The aim of this study was to investigate the effect of initial acidity of wastewater on performance of sulfate-reducing biocathodes. MECs with biocathodes were operated with initial pH values of catholyte ranged from 3.0 to 7.0. The optimum initial pH value was 6.0 with a maximum sulfate reductive rate and biomass of 57 mg L⁻¹ d⁻¹ and 2.1 ± 0.4 mg g⁻¹, respectively. With initial pH 7.0, the pH value of catholyte increased to 9.8 ± 0.2 after an operation cycle, which resulted in low performance of the biocathode. A considerable sulfate reductive rate of 31 ± 0.85 mg L⁻¹ d⁻¹ was achieved with initial pH 3.0. Desulfovibrio sp. grew dominantly with abundance of 46%–66% in the cathode biofilm with initial pH values from 3.0 to 6.0 and contributed to the sulfate reduction. Clostridium and Parapedobacter also had high abundance in pH 6.0 cathode, indicated that interspecies electron transfer between electrochemical active and sulfate-reducing bacteria could play an important role in sulfate removal. The results suggest that acidity of catholyte is an important factor to be considered to utilize autotrophic biocathode MECs for wastewater treatment.     

Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria,erythrocytes and tumor cells

The host-defense peptide, esculentin-2CHa (GFSSIFRGVA¹⁰KFASKGLGK D²⁰LAKLGVDLVA³⁰ CKISKQC) shows potent (MIC ≤ 6 μM) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC₅₀ = 150 μM) and human non-small cell lung adenocarcinoma A549 cells (LC₅₀ = 10 μM). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-α production by peritoneal macrophages. Effects on IL-6 and IL-1β production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys³¹ and Cys³⁷ residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC₅₀ = 11 μM) and A549 cells (LC₅₀ = 3 μM).     

Rotating discs bioreactor,a new tool for lipopeptides production

Microbial production of two biosurfactants, fengycin and surfactin, by Bacillus subtilis ATCC 21332 in a rotating discs bioreactor was studied. Simultaneous production of these lipopeptides was performed by free and cells immobilized on the surfaces of rotating discs. The aeration applied on surface allowed a non-foaming fermentation process and an important production of lipopeptides for low microbial growth in the culture medium. It was demonstrated that the selectivity of lipopeptides synthesis could be modified varying operating conditions and that the cells immobilization improved greatly fengycin synthesis. The maximal concentration of fengycin and surfactin obtained were 838 mg L^?1 and 212 mg L^?1, respectively. The development of this bubble-less process could advance the scale-up of the fermenters for production of biosurfactants.     

Improved accumulation of phenylethanoid glycosides by precursor feeding to suspension culture of Cistanche salsa

Effects of some precursors on phenylethanoid glycosides (PeGs) accumulation in Cistanche salsa cell suspension cultures were investigated. Precursors such as tyrosine, phenylalanine, caffeic acid and cucumber juice at proper concentrations could increase the total accumulation of PeGs (echinacoside, acteoside, 2′-acetylacteoside) by 50%, 12%, 12% and 23%, respectively. Under the combined feeding of precursors at proper concentrations, the total production of PeGs in bio-staged culture reached the highest amount of 1358.1 mg l⁻¹ (640.8 mg echinacoside l⁻¹, 689.4 mg acteoside l⁻¹ and 54.9 mg 2′-acetylacteoside l⁻¹), which was about two-fold of that in the control. This study showed promise for obtaining large-scale production of active ingredients in plant cells by the solid–liquid two step culture (SLTSC) technique and also provided for the first time an example for producing PeGs by C. salsa cell culture. The improved production of PeGs was higher than that in previous reports on PeG production by Cistanche deserticola cell culture fed with precursors.     

Optimization of carotenoid production by Rhodotorula graminis DBVPG 7021 as a function of trace element concentration by means of response surface analysis

As the final step of a study aiming at the optimization of culture conditions for the production of carotenoids by red yeasts, a statistically-based experimental design has been applied to assess the influence of selected trace elements on carotenogenesis in Rhodotorula graminis DBVPG 7021. In particular, a central composite design scheme has been used to evaluate the influence of Fe³⁺, Co²⁺, Mn²⁺, Al²⁺ and Zn²⁺ (within the range 0–50 ppm) on various responses, namely biomass (B), total carotenoid production (TC) and percentage of specific carotenoids (β-CAR, β-carotene; γ-CAR, γ-carotene; TN, torulene; TD, torularhodin) on total carotenoids. Second-order polynomial models were calculated and reduced equations were designed by neglecting non-significant (P < 0.01) regression coefficients. Reduced equations were used to calculate the optimal concentration of trace elements in view of maximising the level of B, TC, β-CAR, γ-CAR, TN and TD. After optimization, average final values total carotenoids (TC = 803.2 μg/g DW) was about 370% of value observed as central point of the central composite design scheme. Under the same condition, average final values of other responses were: B = 5.40 g/L; β-CAR = 50.3%; γ-CAR = 15.4%; TN = 22.7%; TD = 11.6%. All above experimental data are in good agreement with calculated ones, thus confirming the reliability of the proposed empirical model in describing carotenoid production by R. graminis as a function of trace element concentrations.     

Zoledronate-activated Vγ9γδ T cell-based immunotherapy is feasible and restores the impairment of γδ T cells in patients with solid tumors

Gamma/delta (γδ) T cells play a role in innate immunity and exhibit cytotoxicity toward a large range of tumor types. Recent studies have shown that aminobisphosphonates may be applied to a culture in which a large number of γδ T cells are proliferated ex vivo. We carried out a clinical study of 25 patients with various solid tumors to determine further the safety, immunologic effect and feasibility of zoledronate-activated Vγ9γδ T cell-based immunotherapy. No severe toxicity was observed. In the cells used for the first treatment, the total cell number, frequency and number of CD3⁺ Vγ9⁺ γδ T cells were 409 ± 284 × 10⁷ cells, 56 ± 33% and 255 ± 242 × 10⁷ cells, respectively. Aminobisphosphonate therapy or chemotherapy resulted in the suppression of CD3⁺ Vγ9⁺ γδ T-cell proliferation. The numbers of CD3⁺ T cells, CD3⁺ Vγ9⁺ γδ T cells and CD27^? CD45RA^? Vγ9⁺ subsets in peripheral blood were significantly lower in patients than in healthy subjects (P <y 0.05). From such an impaired immunologic condition, the numbers and frequencies of CD3⁺ Vγ9⁺ γδ T cells and CD27^? CD45RA^? subsets significantly increased in patients treated with this immunotherapy. Zoledronate-activated Vγ9γδ T cell-based immunotherapy that restores the number of Vγ9γδ T cells in cancer patients may provide another mode of adoptive immunotherapy.     

Biodegradation of Tapis blended crude oil in marine sediment by a consortium of symbiotic bacteria

Biodegradation rate and the high molecular weight hydrocarbons are among the important concerns for bioremediation of crude oil. Inoculation of a non-oil-degrading bacterium as supplementary bacteria increased oil biodegradation from 57.1% to 63.0% after 10 days of incubation. Both the oil-degrading bacteria and the non-oil-degrading bacteria were isolated from Malaysian marine environment. Based on the 16S rDNA sequences, the oil-degrading bacteria was identified as Pseudomonas pseudoalcaligenes (99% similarity) while the non-oil-degrading bacterium was Erythrobacter citreus (99% similarity). E. citreus does not grow on crude oil enriched medium under present experimental condition but it withstands 5000 mg kg^?1 Tapis blended crude oil in sediment. Under optimal condition, the oil-degrading bacterium; P. pseudoalcaligenes, alone utilized 583.3 ± 3.8 mg kg^?1 (57.1%) at the rate of 3.97 × 10^?10 mg kg^?1 cell^?1 day^?1 Tapis blended crude oil from 1000 mg kg^?1 oil-contaminated sediment. Inoculation of E. citreus as the supplementary bacteria to P. pseudoalcaligenes enhanced biodegradation. The bacterial consortium degraded 675.8 ± 18.5 mg kg^?1 (63.0%) Tapis blended crude oil from the 1000 mg kg^?1 oil-contaminated sediment. Biodegradation rate of the bacterial consortium increased significantly to 4.59 × 10^?10 mg kg^?1 cell^?1 day^?1 (p = 0.02). Improvement of the oil degradation by the bacterial consortium was due to the synergetic reaction among the bacterial inoculants. There are two implications: (1) E. citreus may have a role in removing self-growth-inhibiting compounds of P. pseudoalcaligens. (2) P. pseudoalcaligenes degraded Tapis blended crude oil while E. citreus competes for the partially degraded hydrocarbons by P. pseudoalcaligenes. P. pseudoalcaligenes forced to breakdown more hydrocarbons to sustain its metabolic requirement. The bacterial consortium degraded 78.7% of (C₁₂–C₃₄) total aliphatic hydrocarbons (TAHs) and 74.1% of the 16 USEPA prioritized polycyclic aromatic hydrocarbons.     

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO₂ assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100 μmol photons m⁻² s⁻¹ light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100 μmol photons m⁻² s⁻¹ light condition. Under 15 μmol photons m⁻² s⁻¹ light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.     

Wall shear over high degree stenoses pertinent to atherothrombosis

Atherothrombosis can induce acute myocardial infarction and stroke by progressive stenosis of a blood vessel lumen to full occlusion. Since thrombus formation and embolization may be shear-dependent, we quantify the magnitude of shear rates in idealized severely stenotic coronary arteries (≥75% by diameter) using computational fluid dynamics to characterize the shear environment that may exist during atherothrombosis. Maximum shear rates in severe short stenoses were found to exceed 250,000 s^?1 (9500 dynes/cm²) and can reach a peak value of 425,000 s^?1 for a 98% stenosis. These high shear rates exceed typical shear used for in vitro blood flow experiments by an order of magnitude, indicating the need to examine thrombosis at very high shear rates. Pulsatility and stenosis eccentricity were found to have minor effects on the maximum wall shear rates in severe stenoses. In contrast, increases in the stenosis length reduced the maximum shear to 107,000 s^?1 (98% stenosis), while surface roughness could increase focal wall shear rates to a value reaching 610,000 s^?1 (90% stenosis). The “shear histories” of circulating platelets in these stenoses are far below reported activation thresholds. Platelets may be required to form bonds in 5 μs and resist shear forces reaching 8000 pN per platelet. Arterial thrombosis occurs in the face of pathological high shear stress, creating rapid and strong bonds without prior activation of circulating platelets.     

Prevention of cell death by the zinc ion chelating agent TPEN in cultured PC12 cells exposed to Oxygen–Glucose Deprivation (OGD)

To elucidate the role of Zn²⁺-associated glutamate signaling pathway and voltage-dependent outward potassium ion currents in neuronal death induced by hypoxia–ischemia, PC12 cells were exposed to Oxygen–Glucose Deprivation (OGD) solution mimicking the hypoxic–ischemic condition in neuron, and the effect of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn²⁺ chelating agent on OGD-induced neuronal death was assessed in the present study. The cell survival rate, apoptosis status, potassium channel currents, intracellular free glutamate concentration and GluR2 expression in PC12 cells exposed to OGD in the absence or presence of TPEN for different time were investigated. The results showed that OGD exposure increased apoptosis, reduced the cell viability (P < 0.01 at 3 h, 6 h and 24 h, respectively compared to control), changed the voltage-dependent outward potassium ion current (increase at 1 h, but decrease at 3 h) and decreased the concentration of intracellular glutamate (P < 0.05 at 3 h and 6 h, P < 0.01 at 24 h respectively compared to control) and GluR2 expression (P < 0.05 at 3 h, 6 h and 24 h, respectively compared to control) in PC12 cells. TPEN partially reversed the influence resulted from OGD. These results suggest that OGD-induced cell apoptosis and/or death is mediated by the alteration in glutamate signaling pathway and the voltage-dependent outward potassium ion currents, while TPEN effectively prevent cell apoptosis and/or death under hypoxic–ischemic condition.     

Improvement in natamycin production by Streptomyces natalensis with the addition of short-chain carboxylic acids

Natamycin is an important tetraene (polyene) antibiotic produced in submerged culture by different strains of Streptomyces sp. In the present work, the effects of the addition of short-chain carboxylic acids (acetic, propionic and butyric) on cell growth and the kinetics of natamycin production were investigated during submerged cultivation of Streptomyces natalensis. The addition of acetic and propionic acids showed stimulatory effects on natamycin production when added to the fermentation medium at concentrations below 2 g L^?1 at the beginning of cultivation. In addition, when acetic and propionic acids were added in a mixture (7:1) at a total concentration of 2 g L^?1, antibiotic production increased significantly, reaching 3.0 g L^?1 (approximately 223% and 250% increases in volumetric and specific antibiotic production, respectively, compared with the control culture). Moreover, the addition of carboxylic acids not only increased the antibiotic yield but also decreased the production time from 96 h to only 84 h in shake-flask cultures. A further enhancement in natamycin production was achieved by cultivation in a 2-L stirred-tank bioreactor under controlled pH conditions. The maximum volumetric production of 3.98 g L^?1 was achieved after 84 h in carboxylic acid-supplemented culture (acetate and propionate in a ratio of 7:1).