Addition Of Substrate Research Articles

Effects of sugarcane substrate inputs on microbial biomass and nitrogen availability in tropical sandy soils of northeast Thailand

ABSTRACT The increasing demand for sugarcane residue to produce industrial biofuels competes with organic-matter inputs to tropical soils for increasing fertility. Soil nitrogen (N) availability to plants and carbon (C) storage can be optimized by the selection of substrate types and quantities. We tested whether substrate-induced microbial growth increases soil N mineralization and C stabilization as substrate inputs increase. The effects of substrate additions (cane sugar, leaf litter, and humic acid extracted from filter cake) on microbial growth and N availability were studied using C3/C4 plant C isotopic signatures in a sandy soil of northeast Thailand. The initial growth of microbial biomass in cane sugar-amended soil caused a short-term flush of N derived from the native soil organic matter within 3 days, whereas in the soils amended with leaf litter or humic acid, N was retained in microbial biomass or dissolved organic matter, respectively. Net mineralization of the native soil organic N was maximized by sugar addition at a dosage corresponding to 10 folds of microbial biomass-C. The efficacy of substrates stabilized in the heavy density fraction (>1.6 g cm−3) followed the order: leaf litter > humic acid > sugar. Soil net N mineralization can be promoted by sugar inputs (e.g., bud node) at an early growth stage, while leaf litter and humic acid inputs can increase C storage efficiently in the sandy soil without increasing risks of N starvation and loss when they are applied at several tons of C per hectare at least 3 weeks before planting.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

The Destruction of Cytochrome c in a Complex with Cardiolipin during Catalysis of Lipid Peroxidation

The destruction of cytochrome c during its catalysis of the lipoperoxidase reaction, which causes the impairment of mitochondrial membranes in living cells and the release of various pro-apoptotic factors into the cytosol, was studied. Spectrophotometric analysis showed a much more intense destruction of cytochrome c when hydrogen peroxide was added in the presence of tetraoleylcardiolipin at protein : cardiolipin ratios of 1 : 30 and 1 : 60, which were optimal for the formation of a cytochrome c complex with cardiolipin, compared to the sample in which only hydrogen peroxide and cytochrome c were present. In the second case, the destruction of the porphyrite group of heme took the form of a linear function, while in the presence of cardiolipin the dependence was clearly exponential; upon addition of a lipid substrate, phosphatidic acid, the value of the first-order rate constant of the cytochrome c destruction increased. It is proposed that the rapid destruction of cytochrome c during its catalysis of the lipoperoxidase reaction is an evolutionarily developed mechanism for preventing the spontaneous initiation of apoptosis.

Evaluation of a nitrite-free commercial preparation in the production of swine and roe deer (Capreolus capreolus) salami

The aim of this study was to compare the effect of a commercial formulation consisting of natural ingredients and dextrose on the production of salami. We analysed the effect on microbiological, physico-chemical and sensorial properties in the production of nitrite-free swine and roe deer (Capreolus capreolus) salami. Six batches of salami were prepared: four with the addition of starter cultures and diverse substrates (no substrate, skimmed milk, commercial formulation and dextrose) and two without starter cultures as controls (one without substrate and one with skimmed milk). Samples for microbiological and physico-chemical analysis were taken on day 0, day 5 and day 25. A sensory evaluation was performed at the end of the ripening process. The best results regarding the changes in the microbiota were observed in the batch produced with the addition of skimmed milk (final concentration for Pseudomonas spp. was 3.07 ± 1.11 log cfu g−1, for Enterobacteriaceae was 1.57 ± 0.51 log cfu g−1 and the total coliforms were undetectable). As regards the sensory evaluation, the best scores (in particular colour uniformity, fat/lean distribution and mould flavour) were assigned to the salami produced with the addition of dextrose. According to the results obtained in this study, the addition of the commercial formulation to manufacture nitrite and nitrate-free, dry-cured swine and roe deer salami led to no significant effects on their microbiological and sensory characteristics. Overall, the results obtained with the addition of the commercial formulation were very similar to those obtained by adding dextrose, which is the main component of the formulation itself. HIGHLIGHTS The effect of a commercial formulation on microbiological, chemical and sensory characteristics of nitrite-free swine and roe deer (Capreolus capreolus) salami was analysed. The results obtained with the addition of the commercial formulation were very similar to those obtained by simply adding dextrose or just skimmed milk. The use of commercial preparation seems therefore not worthwhile.

Arene‐Immobilized Ru(II)/TsDPEN Complexes: Synthesis and Applications to the Asymmetric Transfer Hydrogenation of Ketones

AbstractThe Noyori‐Ikariya (arene)Ru(II)/TsDPEN precatalyst has been anchored to amorphous silica and DAVISIL through the η6‐coordinated arene ligand via a straightforward synthesis and the derived systems, (arene)Ru(II)/TsDPEN@silica and (arene)Ru(II)/TsDPEN@DAVISIL, form highly efficient catalysts for the asymmetric transfer hydrogenation of a range of electron‐rich and electron‐poor aromatic ketones, giving good conversion and excellent ee's under mild reaction conditions. Moreover, catalyst generated in situ immediately prior to addition of substrate and hydrogen donor, by reaction of silica‐supported [(arene)RuCl2]2 with (S,S)‐TsDPEN, was as efficient as that generated from its preformed counterpart [(arene)Ru{(S,S)‐TsDPEN}Cl]@silica. Gratifyingly, the initial TOFs (up to 1085 h−1) and ee's (96–97 %) obtained with these catalysts either rivalled or outperformed those previously reported for catalysts supported by either silica or polymer immobilized through one of the nitrogen atoms of TsDPEN. While the high ee's were also maintained during recycle studies, the conversion dropped steadily over the first three runs due to gradual leaching of the ruthenium.

Microalgae production using photo-bioreactor with intermittent aeration for municipal wastewater substrate and nutrient removal

Microalgae has emerged as a promising approach for removing substrate and nutrient from wastewater with the concomitant biofuel production. The substrate and nutrient removal are influenced by several factors such as C/N ratio, F/M ratio, pH, and DO. This study aims to determine the efficiency of substrate and nutrient removal with the growth rates of microalgae and biomass by varying the addition of aeration and substrate. Intermittently aerated reactors were used with the flow rate of 14 L/minute. The batch reactors were prepared by adding glucose substrate of 50 mg/L (Ra0A), 100 mg/L (Ra0B), and 150 mg/L (Ra0C) without aeration; 50 mg/L (Ra12A), 100 mg/L (Ra12B), and 150 mg/L (Ra12C) with 12-hour aeration, and 50 mg/L (Ra24A), 100 mg/L (Ra24B), and 150 mg/L (Ra24C) with 24-hour aeration. The substrate removal, expressed as chemical oxygen demand (COD) in the reactor with aeration, showed the efficiency of 73.88% ± 2.05 (12-hour aeration), 75.2% ± 3.97 (24hours aeration), and 69.86% ± 5.69(without aeration). Nutrient removal as ammonia-N (NH3-N) gave high removal value of 98.3% ± 0.11 and the removal of nutrient as phosphate (PO3-4) showed the efficiency of 54.3% ± 0.1. The growth rate of microalgae and biomass exhibited the highest value in Ra24C reactor with the values ??of 0.0229/day and 0.1295/day, respectively. The pH values ??indicated a shift from normal to alkaline while DO values ??increased by the addition of 12 and 24-hour aeration.

Structural and Molecular Dynamics of Mycobacterium tuberculosis Malic Enzyme, a Potential Anti-TB Drug Target.

Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ contributes to lipid biosynthesis by providing reductants in the form of NAD(P)H. Here, we present the X-ray crystal structure of MEZ to 3.6 Å. We use biochemical assays to demonstrate MEZ is dimeric in solution and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually flexible, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that dimeric MEZ subunits alternate between open and closed states, and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb and not human malic enzyme homologues.

Factors influencing urea use by giant kelp (Macrocystis pyrifera, Phaeophyceae)

AbstractUrea is an available and readily used source of nitrogen for giant kelp, Macrocystis pyrifera, but little is known about its potential importance for sustaining growth. Results of kinetic experiments indicate urea uptake saturates at an average maximum rate (Vmax) of 0.73–0.92 μmol N g dw−1 h−1 with a half saturation constant (Ks) of 1.02–1.08 μM. The affinity of giant kelp for urea was high relative to that reported for other seaweeds. However, results of similar kinetics experiments with natural, co‐occurring phytoplankton communities indicate that the rate of urea uptake by phytoplankton was > 10‐fold higher than that of giant kelp. Urea uptake by giant kelp decreased 3–12% in darkness (relative to in light) compared to a 66–85% decline for phytoplankton. Similar differences were observed for ammonium and nitrate, suggesting that light intensity and photocycles influence the outcome of competition for N between giant kelp and phytoplankton. Monthly measures of urease in kelp tissues revealed persistent activity at levels that were 100‐fold higher than rates of urea uptake (0.13–0.35 μmol N g fw−1 min−1). This finding, coupled with unsuccessful efforts to induce additional urease activity through substrate additions, suggests that urease plays a role in giant kelp physiology beyond that of processing urea taken up from the environment. Collectively, our results suggest giant kelp uses multiple forms of N including urea to sustain year‐round growth. Its consistent capacity to acquire N during both day and night may help offset its low uptake rates relative to phytoplankton and increase its ability to compete for N during periods of low N availability.

Effect of organic matter concentration and characteristics on mercury mobilization and methylmercury production at an abandoned mine site.

Thousands of abandoned mines throughout the western region of North America contain elevated total-mercury (THg) concentrations. Mercury is mobilized from these sites primarily due to erosion of particulate-bound Hg (THg-P). Organic matter-based soil amendments can promote vegetation growth on mine tailings, reducing erosion and subsequent loading of THg-P into downstream waterbodies. However, the introduction of a labile carbon source may stimulate microbial activity that can produce methylmercury (MeHg)-the more toxic and bioaccumulative form of Hg. Our objectives were to investigate how additions of different organic matter substrates impact Hg mobilization and methylation using a combination of field observations and controlled experiments. Field measurements of water, sediment, and porewater were collected downstream of the site and multi-year monitoring (and load calculations) were conducted at a downstream gaging station. MeHg production was assessed using stable isotope methylation assays and mesocosm experiments that were conducted using different types of organic carbon soil amendments mixed with materials from the mine site. The results showed that >80% of the THg mobilized from the mine was bound to particles and that >90% of the annual Hg loading occurred during the period of elevated discharge during spring snowmelt. Methylation rates varied between different types of soil amendments and were correlated with the components of excitation emission matrices (EEMs) associated with humic acid fractions of organic matter. The mesocosm experiments showed that under anoxic conditions carbon amendments to tailings could significantly increase porewater MeHg concentrations (up to 13±3ng/L). In addition, the carbon amendments significantly increased THg partitioning into porewater. Overall, these results indicate that soil amendment applications to reduce surface erosion at abandoned mine sites could be effective at reducing particulate Hg mobilization to downstream waterbodies; however, some types of carbon amendments can significantly increase Hg methylation as well as increase the mobilization of dissolved THg from the site.

Utilization of low-molecular-weight organic compounds by the filterable fraction of a lotic microbiome.

ABSTRACTFilterable microorganisms participate in dissolved organic carbon (DOC) cycling in freshwater systems, however their exact functional role remains unknown. We determined the taxonomic identity and community dynamics of prokaryotic microbiomes in the 0.22 µm-filtered fraction and unfiltered freshwater from the Conwy River (North Wales, UK) in microcosms and, using targeted metabolomics and 14C-labelling, examined their role in the utilization of amino acids, organic acids and sugars spiked at environmentally-relevant (nanomolar) concentrations. To identify changes in community structure, we used 16S rRNA amplicon and shotgun sequencing. Unlike the unfiltered water samples where the consumption of DOC was rapid, the filtered fraction showed a 3-day lag phase before the consumption started. Analysis of functional categories of clusters of orthologous groups of proteins (COGs) showed that COGs associated with energy production increased in number in both fractions with substrate addition. The filtered fraction utilized low-molecular-weight (LMW) DOC at much slower rates than the whole community. Addition of nanomolar concentrations of LMW DOC did not measurably influence the composition of the microbial community nor the rate of consumption across all substrate types in either fraction. We conclude that due to their low activity, filterable microorganisms play a minor role in LMW DOC processing within a short residence time of lotic freshwater systems.

The effects of metabolic substrates glucose, pyruvate, and lactate added to a skim milk-based semen extender for cooled storage of stallion sperm

Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.8 mM; Pyr-2, Pyr-19), lactate (2 mM, 19.8 mM; Lac-2, Lac-19, respectively), or their combinations (G/P/L-2 or G/P/L-19, respectively) were added to a milk-based extender and their effects were determined on motion characteristics, viability/acrosomal intactness (VAI), lipid peroxidation status (VLPP), and DNA integrity (COMPα-t) of sperm incubated for 1 h at 37 °C, or sperm stored for 24 h at either 10 or 20 °C. At any period and temperature tested, Glu-40, G/P/L-2, and G/P-L-19 resulted in similar motion characteristics (P > 0.05) but were higher than that of other treatment groups (P < 0.05). Mean VAI was highest in Glu-40 (P < 0.05). Mean VLPP was highest in G/P/L-2 and G/P/L-19 groups (P < 0.05), and mean COMPα-t was lowest in Control, Glu-40, G/P/L-2 and G/P/L-19 groups (P < 0.05). All measures of sperm quality were higher in semen stored at 10 °C than 20 °C (P < 0.05). In Experiment 2, increasing concentrations of either pyruvate or lactate (Pyr-40, Lac-40 or Pyr-80, Lac-80) were added to the extender as energy substrates and compared to glucose (40 mM), following storage for 72 h at either 10 or 20 °C. Groups Glu-40 and Pyr-40 yielded similar sperm motion characteristics and VAI, while VLPP and COMPα-t were reduced in these treatment groups, as compared to Pyr-80, Lac-40, and Lac-80 (P < 0.05). All measures of sperm quality were higher in semen stored at 10 °C vs 20 °C (P < 0.05). This study demonstrates that at storage temperatures of 10 or 20 °C, stallion sperm quality is optimized by the presence of glucose in a skim milk-based semen extender. The addition of substrates that readily support oxidative phosphorylation (i.e., pyruvate or lactate) did not improve the quality of stallion sperm over that of glucose alone and resulted in deleterious effects on sperm quality over time. These effects appeared to be associated with oxidative stress. Use of pyruvate (40 mM) as an alternative energy substrate to glucose generally yielded similar results to that of glucose when sperm were stored at 10 °C only.

Induction of Fusarium lytic Enzymes by Extracts from Resistant and Susceptible Cultivars of Pea (Pisum sativum L.).

Being pathogenic fungi, Fusarium produce various extracellular cell wall-degrading enzymes (CWDEs) that degrade the polysaccharides in the plant cell wall. They also produce mycotoxins that contaminate grains, thereby posing a serious threat to animals and human beings. Exposure to mycotoxins occurs through ingestion of contaminated grains, inhalation and through skin absorption, thereby causing mycotoxicoses. The toxins weaken the host plant, allowing the pathogen to invade successfully, with the efficiency varying from strain to strain and depending on the plant infected. Fusarium oxysporum predominantly produces moniliformin and cyclodepsipeptides, whereas F. proliferatum produces fumonisins. The aim of the study was to understand the role of various substrates and pea plant extracts in inducing the production of CWDEs and mycotoxins. Additionally, to monitor the differences in their levels when susceptible and resistant pea plant extracts were supplemented. The cultures of F. proliferatum and F. oxysporum strains were supplemented with various potential inducers of CWDEs. During the initial days after the addition of substrates, the fungus cocultivated with pea extracts and other carbon substrates showed increased activities of β-glucosidase, xylanase, exo-1,4-glucanase and lipase. The highest inhibition of mycelium growth (57%) was found in the cultures of F. proliferatum strain PEA1 upon the addition of cv. Sokolik extract. The lowest fumonisin content was exhibited by the cultures with the pea extracts and oat bran added, and this can be related to the secondary metabolites and antioxidants present in these substrates.

Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved pCO2, and requires exogenous vitamin B12. In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (∼5.6 µM) O2 concentrations. The abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb3-type terminal oxidase increased under O2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O2-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

Fabrication of chitosan-polyvinyl alcohol and silk electrospun fiber seeded with differentiated keratinocyte for skin tissue regeneration in animal wound model

Hybrid fibrous mat containing cell interactive molecules offers the ability to deliver the cells and drugs in wound bed, which will help to achieve a high therapeutic treatment. In this study, a co-electrospun hybrid of polyvinyl alcohol (PVA), chitosan (Ch) and silk fibrous mat was developed and their wound healing potential by localizing bone marrow mesenchymal stem cells (MSCs)-derived keratinocytes on it was evaluated in vitro and in vivo. It was expected that fabricated hybrid construct could promote wound healing due to its structure, physical, biological specifications. The fabricated fibrous mats were characterized for their structural, mechanical and biochemical properties. The shape uniformity and pore size of fibers showed smooth and homogenous structures of them. Fourier transform infrared spectroscopy (FTIR) verified all typical absorption characteristics of Ch-PVA + Silk polymers as well as Ch-PVA or pure PVA substrates. The contact angle and wettability measurement of fibers showed that mats found moderate hydrophilicity by addition of Ch and silk substrates compared with PVA alone. The mechanical features of Ch-PVA + Silk fibrous mat increase significantly through co-electrospun process as well as hybridization of these synthetic and natural polymers. Higher degrees of cellular attachment and proliferation obtained on Ch-PVA + Silk fibers compared with PVA and Ch-PVA fibers. In terms of the capability of Ch-PVA + Silk fibers and MSC-derived keratinocytes, histological analysis and skin regeneration results showed this novel fibrous construct could be suggested as a skin substitute in the repair of injured skin and regenerative medicine applications.

Assessing microbial residues in soil as a potential carbon sink and moderator of carbon use efficiency

A longstanding assumption of glucose tracing experiments is that all glucose is microbially utilized during short incubations of ≤2 days to become microbial biomass or carbon dioxide. Carbon use efficiency (CUE) estimates have consequently ignored the formation of residues (non-living microbial products) although such materials could represent an important sink of glucose that is prone to stabilization as soil organic matter. We examined the dynamics of microbial residue formation from a short tracer experiment with frequent samplings over 72 h, and conducted a meta-analysis of previously published glucose tracing studies to assess the generality of these experimental results. Both our experiment and meta-analysis indicated 30–34% of amended glucose-C (13C or 14C) was in the form of residues within the first 6 h of substrate addition. We expand the conventional efficiency calculation to include residues in both the numerator and denominator of efficiency, thereby deriving a novel metric of the potential persistence of glucose-C in soil as living microbial biomass plus residues (‘carbon stabilization efficiency’). This new metric indicates nearly 40% of amended glucose-C persists in soil 180 days after amendment, the majority as non-biomass residues. Starting microbial biomass and clay content emerge as critical factors that positively promote such long term stabilization of labile C. Rapid residue production supports the conclusion that non-growth maintenance activity can illicit high demands for C in soil, perhaps equaling that directed towards growth, and that residues may have an underestimated role in the cycling and sequestration potential of C in soil.

Palladium-Catalyzed Anti-Markovnikov Oxidation of Aromatic and Aliphatic Alkenes to Terminal Acetals and Aldehydes

AbstractCatalytic anti-Markovnikov (AM) oxidation of terminal alkenes can provide terminally oxyfunctionalized organic compounds. This short review mainly summarizes our recent progress on the Pd-catalyzed AM oxidations of aromatic and aliphatic terminal alkenes to give terminal acetals (oxidative acetalization) and aldehydes (Wacker-type oxidation), along with related reports. These reactions demonstrate the efficacy of the PdCl2(MeCN)2/CuCl/electron-deficient cyclic alkenes/O2 catalytic system. Notably, electron-deficient cyclic alkenes such as p-benzoquinones (BQs) and maleimides are key additives that facilitate nucleophilic attack of oxygen nucleophiles on coordinated terminal alkenes and enhance the AM selectivity. BQs also function to oxidize Pd(0) depending on the reaction conditions. Several other factors that improve the AM selectivity, such as the steric demand of the nucleo­philes, slow substrate addition, and halogen-directing groups, are also discussed.1 Introduction2 Anti-Markovnikov Oxidation of Aromatic Alkenes to Terminal Acetals­3 Anti-Markovnikov Oxidation of Aromatic Alkenes to Aldehydes4 Anti-Markovnikov Oxidation of Aliphatic Alkenes to Terminal Acetals­5 Anti-Markovnikov Oxidation of Aliphatic Alkenes to Aldehydes6 Conclusion

Validation of a Modified Anti-FXa Assay on STA-Compact Analyzer for Measuring FXa Inhibitor Levels in the Presence of Andexanet Alfa

Introduction: The anticoagulants rivaroxaban and apixaban inhibit Factor Xa (FXa) activity and are effective therapeutic agents for the prevention and treatment of thromboembolism. Andexanet alfa (andexanet) is the only specific reversal agent approved for anticoagulation reversal in patients presenting with life-threatening bleeding treated with rivaroxaban or apixaban. Anti-FXa assays are functional assays used for measuring direct and indirect FXa inhibitor levels in plasma. Current commercially available anti-FXa assays are not suitable for accurately measuring anti-FXa activity in patient samples in the presence of andexanet due to the high sample dilutions (e.g.,1:44 with STA-Liquid Anti-Xa), which causes dissociation of the inhibitor from andexanet (due to reversible binding). This results in substantial underestimation of the reversal activity of andexanet, and in some cases with erroneously elevated anti-FXa levels in patient samples following andexanet treatment. Therefore, we modified the current anti-FXa assays to measure apixaban and rivaroxaban anti-FXa levels in the presence of andexanet. Methods: The modified anti-FXa assays were performed on STA®-Compact analyzer using reagents from STA-Liquid Anti-Xa assay kits (Stago). Andexanet was provided as a 10 mg/mL frozen stock. Plasma samples with FXa inhibitor in the presence or absence of andexanet were prepared using Stago Calibrator 1 and pooled normal human plasma (Precision Biologic). Previous anti-FXa assays (calibrator range: 0-500 ng/mL) employed 1:4 dilution of test plasma in Owren-Koller buffer followed by addition of FXa substrate and bovine FXa with an overall 1:44 sample dilution. In the modified assays, calibration curves for the anti-FXa assay were generated using apixaban or rivaroxaban calibrators in the range of 0 to 80 ng/mL; test samples were analyzed neat with an overall 1:2.6 sample dilution. Rivaroxaban and apixaban concentrations in the test samples were interpolated from the respective assay specific calibration curves. Validation of the modified anti-FXa assays was performed by assessing a) linearity and precision of the calibration curve and controls, b) lower limit of quantitation (LLOQ), c) inter-assay precision and d) sample stability. Results: The calibration curves for both apixaban and rivaroxaban assays demonstrated good correlation with a mean "r" value of 0.997 and 0.998, respectively (n=6). The percent recovery and precision of the modified anti-FXa assay are summarized in Table 1. The LLOQ for apixaban and rivaroxaban using the modified assays were &amp;lt;18.8 and &amp;lt;20.6 ng/mL, respectively, based on the preliminary results. Both apixaban and rivaroxaban assays demonstrated potent reversal of anti-FXa activity in presence of andexanet. Samples containing 232 ng/mL apixaban (0.5 µM) treated with equimolar andexanet (0.5 µM) resulted in 77.18 ng/mL of quantifiable apixaban (~66.7% reversal). Similarly, samples containing 242 ng/mL rivaroxaban (0.5 µM) treated with equimolar andexanet (0.5 µM) resulted in 29.44 ng/mL quantifiable rivaroxaban (~87.8% reversal). The short-term stability study with the modified anti-FXa assay included storage of plasma samples at 2-8oC up to 24 hours and under frozen conditions at -20oC up to 2 weeks. Conclusions: The modified anti-FXa assays intended for measuring FXa inhibitor levels in the presence of andexanet produced acceptable analytical performance characteristics. Application of the modified anti-FXa assays in plasma samples from andexanet-treated human subjects has yet to be studied. Table 1 Disclosures Cardenas: CirQuest Labs/MLM Medical Labs: Current Employment. Kotha:CirQuest Labs/MLM Medical Labs: Current Employment. Lu:Portola Pharmaceuticals, Inc.: Current Employment. Conley:Portola Pharmaceuticals, Inc.: Current Employment. Bourdin:Diagnostica Stago: Current Employment. Herve:Diagnostica Stago: Current Employment. Jennings:CirQuest Labs/MLM Medical Labs: Current Employment, Current equity holder in private company.

Mechanistic Studies of Thrombin Generation Assay to Evaluate Procoagulant Potential of Fitusiran

Introduction: Fitusiran is a once-monthly subcutaneously administered RNA interference therapeutic targeting antithrombin III (AT) to rebalance hemostasis in patients with hemophilia A and B with or without inhibitors. As for other non-FVIII therapies in development, there is a need to better assess the procoagulant potential of fitusiran. Using thrombin generation assays (TGA), we studied the hemostatic activity in hemA plasma with low AT levels in the presence and absence of varying factor VIII (FVIII) levels to mimic conditions associated with breakthrough bleeds. To better understand the impact of AT depletion in the assay, we monitored multiple TGA parameters as well as the levels of several coagulation components potentially influencing the reactions. Methods: Pooled HemA plasma was applied to an AT affinity resin to remove AT. Residual AT activity was 5% and additional AT was spiked back to produce HemA plasmas with various amounts of AT (5, 10, 20 and 100%). FVIII was spiked into the 4 plasmas at 0, 5, 10, 20, 30, 50, and 100 IU/dL and all samples were analyzed by Calibrated Automated Thrombography (Stago). Thrombin generation parameters were calculated by thrombinoscope software.After the TGA reaction, samples were further analyzed by immunoblot for evaluation of thrombin-AT (TAT) and alpha2-macroglobulin-thrombin (a2M:T) complex formation, as well as remaining (pro)thrombin, AT and a2M levels . In parallel, a2M:T complex levels were assessed by ELISA capture of a2M followed by addition of thrombin substrate to directly measure the thrombin activity stemming from a2M-inhibited thrombin. . Results: TGA parameters for FVIII titrations in HemA plasmas with reduced AT were calculated and compared to the FVIII standard curves generated in HemA plasma with normal AT levels for each TGA parameter (lag time, time to peak, peak height, ETP and velocity index). As expected, a greater reduction of AT activity in hemA plasma correlated with increased thrombin generation compared to hemA plasma (with 100% AT). The apparent FVIII-like activity observed at low AT conditions in HemA plasma was dependent on the TGA parameter evaluated, with velocity index suggesting the lowest and ETP the highest FVIII-like activity. Furthermore, particularly at lower starting levels of AT, AT is depleted during the TGA reaction and leads to enhanced levels of a2M:T complex as observed by immunoblot or a2M:T chromocapture assays. Importantly, the a2M:T complex is capable of cleaving the assay substrate and contributing to the TGA signal. Typically, the a2M:T generated signal is calculated and substracted by the thrombinoscope software. However, under low AT conditions, increased levels of a2M-T lead to substrate consumption at later stages of the reaction, resulting in erroneous underestimation of the a2M:T activity that does not agree with direct measurement of a2M: T levels. These limitations need to be taken into consideration when interpreting TGA data under conditions of low AT activity. While this remains an area of investigation, parameters derived earlier in the reaction prior to AT depletion and significant levels of a2M:T complex formation may better correlate with the true hemostatic potential associated with AT inhibition. Conclusion: Our results show that TGA can be a valuable tool to measure the hemostatic potential associated with fitusiran therapy. We demonstrate that reduced levels of AT are associated with enhanced thrombin generation, but that activity comparison to FVIII varies significantly depending on the TGA parameter analyzed. In addition we show that under low AT conditions, AT can become depleted leading to a2M:T complex accumulation which may lead to overestimation of some TGA parameters. This overestimation is likely an artifact of the TGA method as it is a closed in vitro system that can be influenced by the depletion of components or accumulation of products. Our results suggest that TGA parameters calculated early in the reaction are less influenced by some of these limitations and may be better predictors of the true hemostatic activity of fitusiran. Furthermore, a2M may play a more active role during fitusiran treatment, ensuring free thrombin does not migrate outside of the site of injury. Disclosures Kattula: Sanofi: Current Employment, Current equity holder in publicly-traded company. Salvador:Sanofi: Current Employment, Current equity holder in publicly-traded company. Wang:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. van der Flier:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:Sanofi: Current Employment, Current equity holder in publicly-traded company.

Recycling of Chrysoporthe cubensis enzymes to overcome the adsorption in lignin and to improve sugarcane bagasse saccharification

Biomass enzymatic hydrolysis efficiency shows some limitations, such as the non-specific adsorption of enzymes in lignin. We evaluated the adsorption of proteins and enzymes from Chrysoporthe cubensis into alkaline and acid pretreated sugarcane bagasse, containing 8.1 and 33.8% of lignin, respectively. The data from Langmuir isotherm showed that at 4 °C, proteins were more adsorbed by acid-treated bagasse, 10.5 mg g−1, while higher adsorption affinity, 2.0 mL mg−1, was determined from alkali-treated bagasse. Similarly, under hydrolytic conditions (50 °C), higher amounts of proteins were adsorbed into acid-treated bagasse, confirming the predominant effect of lignin on protein adsorption. Enzymatic recycling rounds during alkali-treated bagasse saccharification showed that the addition of fresh substrate and enzymes promoted the higher release of glucose and xylose, 40.3 and 38.3 g L−1, respectively. However, the addition of only fresh substrates showed to be efficient for C. cubensis enzymes recovery and substrate hydrolysis, which reduces enzyme consumption and costs.

Залежність адаптаційної здатності мітохондрій печінки від способу виділення клітин

When conducting studies on isolated hepatocytes, it is important to obtain cells that retain the functional properties that are characteristic of the whole organ. Increased blood viscosity during liver perfusion, decreased perfusion pressure in blood vessels, and hence hypoxia, are among the factors that may affect the functional state of isolated hepatocytes. The functional state of cells can be estimated by the adaptive capacity of mitochondria, by inducing maximal respiration rate by uncoupling respiration and oxidative phosphorylation due to the addition of FCCP. The research aimed to investigate the adaptive capacity of mitochondria of isolated hepatocytes using in situ and in vitro liver perfusion. Hepatocytes were isolated by the two-staged Seglen method by in situ and in vitro liver perfusion. Isolated hepatocytes, after 15-minute incubation in the medium without addition or with respective oxidative substrate – glutamine, pyruvate, succinate, monomethyl succinate, α-ketoglutarate, dimethyl-α-ketoglutarate (at a concentration of 2 mM) or glucose (10 mM) – were added into the respiratory chamber and FCCP was added in increasing concentrations. It was established that at in situ liver perfusion maximal rate of uncoupled respiration and the optimal concentration of FCCP was higher than at in vitro liver perfusion. Addition of exogenous substrates to a medium increased the respiration rate of hepatocytes. Upon in situ liver perfusion maximal uncoupled respiration rate increased at all causes except glucose, and at in vitro liver perfusion – only when dimethyl-α-ketoglutarate, succinate and monomethyl succinate were used. The optimal concentration of FCCP at in vitro liver perfusion increased due to the addition of glutamine, pyruvate and monomethyl succinate to the medium, and at in situ liver perfusion – only upon glucose oxidation. In both perfusion methods, the highest maximal rate of uncoupled respiration is with the use of monomethyl succinate and the optimal FCCP concentration – upon pyruvate oxidation. Therefore, in situ liver perfusion is better method to obtain stable and metabolically active hepatocytes in support respiratory processes at a high level then in vitro perfusion.