Preincubation Conditions Research Articles

Orthophosphate uptake onto woodchips in denitrifying bioreactors is enhanced by anoxic-(anaerobic-)oxic cycling

Woodchips are widely used as a low-cost and renewable organic carbon source for denitrifying biofilms in passive nutrient removal systems. One limitation of wood-based biofiltration systems is their relatively poor removal of phosphorus (P) from subsurface drainage and stormwaters, necessitating the use of additional filter media when co-treatment of nitrogen (N) and P is required. Here, we show that anoxic-oxic cycling of woodchip media, which enhances nitrate (NO3−) removal by increasing the mobilization of organic carbon from wood, also improves orthophosphate (Pi) uptake onto woodchips. Orthophosphate removal rates in flow-through woodchip columns ranged from 0 to 34.9 μg PO43− L−1 h−1 under continuously-saturated (anoxic) conditions, and increased to 17.5 to 71.9 μg PO43− L−1 h−1 in columns undergoing drying-rewetting (oxic-anoxic) cycles. The highest Pi removal efficiencies were observed in the first 20 h after reactors were re-flooded, and were concurrent with maxima in polyphosphate kinase (ppk) gene expression by the polyphosphate accumulating organisms (PAOs) Accumulibacter spp. and Pseudomonas spp. Batch experiments confirmed that anoxic-anaerobic-oxic pre-incubation conditions led to orthophosphate uptake onto woodchips as high as 74.9 ± 0.8 mg PO43−/kg woodchip, and batch tests with autoclaved woodchips demonstrated that Pi uptake was due to biological processes and not adsorption. NO3− removal in batch tests was also greatest under oxic incubation conditions, attributed to greater carbon availability in hypoxic to anoxic zones in woodchip biofilms. While further research is needed to elucidate the mechanisms controlling enhanced Pi uptake by woodchip biofilms under anoxic-(anaerobic-)oxic cycling, these results suggest a role for enhanced Pi uptake by PAOs in a nature-based system for treatment of nonpoint source nutrients.

Unveiling the mechanism of hesperidin-induced LdTopI-mediated cell death pathway in protozoan parasite Leishmania donovani

Unicellular protozoan parasite Leishmania donovani is the causative agent for visceral leishmaniasis (VL) or Kala-azar, a neglected fatal parasitic disease. The conventional treatment of VL consists of therapeutic agents having several shortcomings such as toxicity, high cost, efficacy variance and increased drug resistance. Therefore, there is a desperate need to develop an effective treatment against the parasite. Previous reports suggested that flavonoids can inhibit the enzyme Leishmania donovani DNA topoisomerase I (LdTopILS). Therefore, for the first time in this present study, we divulge HSP (one of the natural sources of flavonoids), as a potent natural antileishmanial compound with efficacy in BALB/c mice at 20 mg/kg of body weight, inhibits LdTopILS at 97 % of its activity at 160 μM in preincubation condition (competitively). It binds with free enzyme and does not allow it to bind with the substrate DNA. Moreover, HSP does not stabilize DNA-topoisomerase I cleavable complex. Thus, HSP acts a catalytic topoisomerase I inhibitor, which inhibits complete activity by binding with Lys269 and Thr411 of large subunit of the enzyme. On the other hand, HSP induces the topo I-mediated programmed cell death process by the formation of cellular reactive oxygen species, resulting in depolarization of mitochondrial membrane potential, followed by fragmentation of nuclear DNA. Therefore, the present study illuminates a natural flavonoid that in future might be a promising lead for the treatment of VL.

Porcine skeletal muscle typing in histochemical and in-situ RT-PCR analysis

Currently, there are plenty of histochemical methods to classify pig muscle fibers, which confused the naming and classification of muscle fibers. This study aims to analyze the difference and correlation of 6 different histochemical methods and select the most suitable method for muscle fiber classification at the molecular and histomological levels by in-situ RT-PCR and enzyme histochemical methods. Muscle fiber samples, including psoas (PM), semitendinosus (SM) and trapezius muscle (TM), were collected from Large Spotted (LS), Lantang (LT) and Landrace (LR) pigs at their market-ages (LS at 150 d, LT at 210 d, and LR at 150 d). 6 kinds of histochemical methods combining actomyosin adenosine triphosphatase (AM-ATPase) with succinate dehydrogenase (SDH) enzyme were conducted to differentiate fiber types. 2 types of fibers (I and II) were differentiated by acid 2-fibre (2-AC) or alkaline 2-fibre classification(2-AL), 3 types of fibers (βR, αR and αW) by 3-AC or 3-AL, and 4 types of fibers (I, IIa, IIx and IIb) by 4-AC, or 4-AL. Results showed that AC and AL muscle-fiber classification were consistent in reflecting the characteristics of muscle fibers(P > 0.05), but the color of each muscle fiber type was just opposite. AC methods may be superior to AL methods because of their clear staining background, the sensitivity to staining condition. But there were breed differences and tissue specificity in the optimal preincubation condition. The optimal acid preincubation condition for classifying muscle fibers was pH4.30 for LT, while pH 4.35 for the LS and LR pigs. Meanwhile the optimal acid preincubation condition was pH4.35 for PM, while pH4.40 for TM or SM. For further selection from 2, 3, 4-AC, in-situ RT-PCR was applied to detect the mRNA distribution of myosin heavy chain I (MyHC-I). By combining in-situ PCR with enzyme histochemistry methods, MyHC-I gene and its product – Type I fibrocytes were directly located in cells at both molecular level and morphological level. Compared with the cross-sectional area (CSA) of different muscle fibers (i.e. I, II, βR, αR, αW, IIa, IIx and IIb) identified by enzyme histochemistry, it was found that the CSAs with stronger mRNA expression signal of MyHC-Ⅰ were closer to those of the Type I muscle fiber measured by 4-AC enzyme histochemistry (P > 0.05). Therefore, 4-AC may be considered as the most proper muscle typing method to study muscle fiber typing as well as meat quality. And the combination of in-situ RT-PCR and histochemistry may help better understand porcine muscle fiber characteristics and meat quality in pigs.

Differential Preincubation Effects of Nicardipine on OATP1B1- and OATP1B3-Mediated Transport in the Presence and Absence of Protein: Implications in Assessing OATP1B1- and OATP1B3-Mediated Drug-Drug Interactions.

Impaired transport activity of hepatic OATP1B1 and OATP1B3 due to drug-drug interactions (DDIs) often leads to increased systemic exposure to substrate drugs (e.g., lipid-lowering statins). Since dyslipidemia and hypertension frequently coexist, statins are often concurrently used with antihypertensives, including calcium channel blockers (CCBs). OATP1B1/1B3-related DDIs in humans have been reported for several CCBs. To date, the OATP1B1/1B3-mediated DDI potential of CCB nicardipine has not been assessed. The current study was designed to assess the OATP1B1- and OATP1B3-mediated DDI potential of nicardipine using the R-value model, following the US-FDA guidance. IC50 values of nicardipine against OATP1B1 and OATP1B3 were determined in transporter-overexpressing human embryonic kidney 293 cells using [3H]-estradiol 17β-D-glucuronide and [3H]-cholecystokinin-8 as substrates, respectively, with or without nicardipine-preincubation in protein-free Hanks' Balanced Salt Solution (HBSS) or in fetal bovine serum (FBS)-containing culture medium. Preincubation with nicardipine for 30 min in protein-free HBSS buffer produced lower IC50 and higher R-values for both OATP1B1 and OATP1B3 compared to in FBS-containing medium, yielding IC50 values of 0.98 and 1.63 µM and R-values of 1.4 and 1.3 for OATP1B1 and OATP1B3, respectively. The R-values were higher than the US-FDA cut-off value of 1.1, supporting that nicardipine has the potential to cause OATP1B1/3-mediated DDIs. Current studies provide insight into the consideration of optimal preincubation conditions when assessing the OATP1B1/3-mediated DDIs in vitro.

The effect of cerium dioxide nanocrystals on the prooxidant status of rat lung fibroblasts in vitro under γ-irradiation conditions

Redox-active nanocrystals of cerium dioxide (CeO2) are of interest as antioxidants; therefore, it is relevant to study the potential ability of these nanocrystals to correct prooxidative markers in irradiated cell culture in vitro. The purpose of the study was to evaluate the effect of redox-active spherical nanocrystals of cerium dioxide on markers of oxidative stress induced by γ-irradiation in a two-dimensional culture of rat lung fibroblasts. Materials and methods. The study was performed on a monolayer of lung fibroblasts of Wistar rats. Nanocrystals were added to the nutrient medium one hour before irradiation until their final concentration in the medium of 2.5 μg/L. The cells were incubated with nanocrystals for one hour and then irradiated. The radiation dose was 0.75 Gy. The concentration of 8-isoprostane was determined by spectrophotometry. The concentration of free oxygen species and the degree of lipid peroxidation in living cells were determined by fluorimetry. Visualization and measurement of fluorescence intensity were performed by confocal laser scanning microscopy. Results. 3 hours after irradiation under the conditions of pre-incubation with CeO2 nanocrystals, the content of reactive oxygen species, the level of lipid peroxidation, and the content of 8-isoprostane were significantly decreased in rat lung fibroblast culture. At the same time, incubation with cerium dioxide nanocrystals much more effectively reduced the content of reactive oxygen species in the cytoplasm of cells than in their nuclei and nucleoli in particular. Conclusion. Preincubation of rat lung fibroblasts in vitro with СeО2 nanocrystals can significantly reduce the oxidizing effects of ionizing radiation.

Static Model-Based Assessment of OATP1B1-Mediated Drug Interactions with Preincubation-Dependent Inhibitors Based on Inactivation and Recovery Kinetics.

Quantitative assessment of drug-drug interactions (DDIs) via organic anion transporting polypeptide (OATP) 1B1 is one of the key issues in drug development. Although OATP1B1 inhibition exhibits unique characteristics, including preincubation dependence for some inhibitors, a limited approach has been attempted based on the static model that considers such preincubation dependence in the prediction of DDIs via OATP1B1. The present study aimed to establish the prediction of DDIs via OATP1B1 using preincubation-dependent inhibitors based on the static model and incorporating both inactivation and recovery of OATP1B1 activity. Cyclosporine A was selected as a preincubation-dependent inhibitor, as well as five substrates that include probes and pharmaceuticals. The inhibition ratio (R value) calculated on the basis of a conventional static model, considering inhibition of OATP1B1 and contribution ratio of OATP1B1 to the overall hepatic uptake, was much lower than the reported AUC ratio, even when IC50 values were estimated after preincubation conditions. Conversely, the R value that was estimated by considering inactivation and recovery parameters was closer to the AUC ratio. The R value that was calculated assuming the complete contribution of OATP1B1 was much higher than the AUC ratio, avoiding false-negative prediction. The R value estimated by considering inactivation and recovery for another combination of a preincubation-dependent inhibitor, asunaprevir, and substrate drug, rosuvastatin, was also closer to the AUC ratio. Thus, R values calculated based on such OATP1B1 kinetics would be potential alternative indexes for the quantitative prediction of OATP1B1-mediated DDIs using preincubation-dependent inhibitors, although this prediction is affected by estimation of the contribution ratio of substrates. SIGNIFICANCE STATEMENT: Static model-based quantitative prediction of organic anion transporting polypeptide 1B1-mediated drug-drug interactions induced by preincubation-dependent inhibitors was newly proposed to avoid false-negative prediction.

Selective inhibition of CYP2C8 by fisetin and its methylated metabolite, geraldol, in human liver microsomes

Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb–drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC50 shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 μM for fisetin and 11.5 μM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs.

Effect of extracted malva nut gum on reducing high glucose levels by Caco-2 cells

Abstract Mucilage of malva nut seeds has been commercially produced as a functional beverage in many South East Asian countries. The purposes of this research were, first, to analyze chemical compositions of different layers of malva nut seeds. Second objective was to investigate the efficacy and dose response of malva nut gum (MNG) for the reduction of glucose uptake in Caco-2 cells. Third objective was to determine the effect of 30 min pre-incubation of Caco-2 cells with various MNG preparations on the uptake of glucose. Caco-2 cells were incubated in Dulbecco's Modified Eagle's Medium (DMEM) containing (i) different concentrations of glucose (i.e. 5.5, 25 mM), (ii) different sources of glucose (i.e. sucrose, starch), (iii) different types of dietary fiber contained in different preparations of gum extracted from malva nut seeds and guar nuts. The cells were incubated in glucose containing DMEM under two different conditions: simultaneous and 30 min pre-incubation conditions. Scanning electron microscopy showed porous surface of extracted MNG compared to malva nut seed. In 25 mM glucose concentrate containing DMEM, the optimal level of dietary fiber to significantly reduce glucose uptake was 0.25% for malva nut seed, extracted MNG and mixed gum, but 0.5% for guar gum. FT-IR showed additional bands in the mixture of each gum and glucose. The contents of glucose uptake for the cells pre-incubated for 30 min, were 50–85% lower than that of the control. These results demonstrate that MNG is effective in lowering glucose uptake by Caco-2 cells in both low and high carbohydrate concentrations.

Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

Biochemical markers of safety of nano-particles of metals on the model of isolated subcultural fractions of eukaryotes

Unique sizes and a high level of bioavailability allow nanoparticles of metals (NPMe) to come into direct contact with biological systems, with infectious agents, toxins, as well as with different chemical compounds and separate cell structures (proteins, lipids, nucleic acids). Other biological effects, including less toxicity than in microscopic substances, require attention to be paid to the study of the potential risk of using nanoparticles of each type in a particular way, therefore scientific support is absolutely necessary in this direction. It is believed that the cytotoxicity of nanomaterials is due to genomic and mutagenic effects, but the mechanical forces of interaction of NPMе with cells, obviously, will change not only cytological but also their metabolic reactions. Therefore, the purpose of this research was to determine the biochemical markers of safety (potential toxicity) of NPMe (Au, Ag, Cu, Fe, Co, GFCo, Zn, MnO2) on the model of isolated membrane and cytosolic fractions of eukaryotic test cells of CHO-K1 and U937 lines. Under conditions of preincubation of experimental samples of NPMe at a final concentration of 1 μg/cm3 by the metal with preparations of subcellular fractions of CHO-K1 and U937 (in the final amount of protein 150–200 μg/cm3) for 3 minutes at 37 ± 1 ºС, there was determined the magnitude of membrane ATP-ase and cytosolic LDH-ase activity compared to intact cells ("control"). According to the results of the research, colloidal dispersions of NPAg average size ~30 nm, NPFe ~100 nm, NPCu ~70 nm, and NPMnO2 ~50 nm are safe and biocompatible by their membranotropic effect on subcellular fractions of eukaryotic test cells, as evidenced by an increase in the level of membrane ATPase and cytosolic LDHase of test-cells CHO-K1, and the experimental samples NPCo, NPGFCo and NPZn average size of ~100 nm are membrane-toxic, that is, dangerous. By the nature of the changes in the enzymatic activity of the test cells U937, the discrete dimensions of the membranotropic action of NPAu have been demonstrated: nanoparticles of size ~10 nm caused the inhibition of the membrane Na+,K+-ATPase, and the size of ~30 nm and ~45 nm – its induction; nanoparticles of size ~10, ~20 and ~30 nm induced cytosolic LDHase and the size of ~45 nm – its inhibition relative to the control level of enzymes, so NPAu ~10 and ~45 nm can be considered membrane toxic, and size ~30 nm – safe and biocompatible for eukaryotic cells. Based on the hypothesis about the involvement of metabolism-dependent mechanisms of contact interaction of colloidal dispersions of experimental samples of NPMe with cells through membranotropic properties, the study of their potential danger or biocompatibility in further research can be carried out by determining the intensity of oxidation of the main structural components of biomembranes of cells – lipids and proteins and indicators of their AO-regulation.

Altered embryonic development in northern bobwhite quail (Colinus virginianus) induced by pre-incubation oscillatory thermal stresses mimicking global warming predictions.

Global warming is likely to alter reproductive success of ground-nesting birds that lay eggs normally left unattended for days or even weeks before actual parental incubation, especially in already warm climates. The native North American bobwhite quail (Colinus virginianus) is such a species, and pre-incubation quail eggs may experience temperatures ≥45°C. Yet, almost nothing is known about embryonic survival after such high pre-incubation temperatures. Freshly laid bobwhite quail eggs were exposed during a 12 day pre-incubation period to one of five thermal regimes: low oscillating temperatures (25–40°C, mean = 28.9°C), high oscillating temperatures (30–45°C, mean = 33.9°C), low constant temperatures (28.85°C), high constant temperatures (mean = 33.9°C), or commercially employed pre-incubation temperatures (20°C). After treatment, eggs were then incubated at a standard 37.5°C to determine subsequent effects on embryonic development rate, survival, water loss, hatching, and embryonic oxygen consumption. Both quantity of heating degree hours during pre-incubation and specific thermal regime (oscillating vs. non-oscillating) profoundly affected important aspects of embryo survival and indices of development and growth Pre-incubation quail eggs showed a remarkable tolerance to constant high temperatures (up to 45°C), surviving for 4.5±0.3 days of subsequent incubation, but high oscillating pre-incubation temperature increased embryo survival (mean survival 12.2±1.8 days) and led to more rapid development than high constant temperature (maximum 38.5°C), even though both groups experienced the same total heating degree-hours. Oxygen consumption was ~200–300 μl O2.egg.min-1 at hatching in all groups, and was not affected by pre-incubation conditions. Oscillating temperatures, which are the norm for pre-incubation quail eggs in their natural habitat, thus enhanced survival at higher temperatures. However, a 5°C increase in pre-incubation temperature, which equates to the predicted long-term increases of 5°C or more, nonetheless reduced hatching rate by approximately 50%. Thus, while pre-incubation bobwhite eggs may be resiliant to moderate oscillating temperature increases, global warming will likely severely impact wild bobwhite quail populations, especially in their strongholds in southern latitudes.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139.

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL−1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL−1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

Effect of pre-incubation and incubation conditions on hatchability and chick quality of Kuroiler chickens

A study was conducted to review the effects of pre-incubation and incubation conditions on hatchability and chick quality of Kuroiler chickens. The most important incubation factors that affect hatch time, hatchability and hatch window (temperature, humidity, turning, and ventilation) were reviewed. There was a significant influence (P<0.05) of breeder age on hatchability. Also, there was significance (P<0.05) of breeder age on texture of the egg shell; while there was no significance (P > 0.05) between size of egg and hatch window. The performance of Kurolilers at the end of the rearing period depended, in part, on the quality of day-old chicks at placement. The quality of day-old chicks was highly affected by the incubation conditions, hatch time (which determines the time spent in the hatchery under high temperature and humidity), and handling of chicks after hatch.Key words: Egg shell, hatch handling, hatch time, hatch window

In vitro inhibition of human cytochrome P450 by cudratricusxanthone A

Cudratricusxanthone A (CTXA) isolated from the roots of Cudrania tricuspidata Bureau (Moraceae) has several biological activities, including hepatoprotective, neuroprotective, anti-inflammatory, monoamine oxidase inhibitory, and antithrombotic activities. In this study, we investigated the potential herb–drug interaction of CTXA and nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) using a cocktail probe assay. CTXA reversibly inhibited the CYP1A2-catalyzed phenacetin O-deethylation, CYP2C8-catalyzed paclitaxel 6-hydroxylation, and CYP2C9-catalyzed diclofenac 4′-hydroxylation with half-maximal inhibitory concentration (IC50) values of 3.9, 4.7, and 2.9 µM, respectively. The IC50 values did not change under different preincubation conditions. CTXA showed marked dose-dependent, but not time-dependent, inhibition of CYP1A2 and 2C9 activities in HLMs. Dixon plots showed typical competitive inhibition of CYP1A2 and CYP2C9 with Ki values of 1.3 and 1.5 µM, respectively. Further, CTXA inhibited CYP2C8 in a non-competitive manner with a Ki value of 2.2 µM. Our results showed that CTXA reversibly inhibits CYP1A2, 2C8, and 2C9.

Mycelial actinobacteria in salt-affected soils of arid territories of Ukraine and Russia

A high population density (up to hundreds of thousands or millions CFU/g soil) of mycelial bacteria (actinomycetes) is determined in salt-affected soils of arid territories of Ukraine, Russia, and Turkmenistan. Of all the studied soils, the lowest amounts of actinomycetes (thousands and tens of thousands CFU/g soil) are isolated from sor (playa) and soda solonchaks developed on the bottoms of drying salt lakes in Buryatia and in the Amu Darya Delta. Actinomycetes of the Streptomyces, Micromonospora, and Nocardiopsis genera were recorded in the studied soils. It is found that conditions of preincubation greatly affect the activity of substrate consumption by the cultures of actinomycetes. This could be attributed to changes in the metabolism of actinomycetes as a mechanism of their adaptation to the increased osmotic pressure of the medium. The alkali tolerance of halotolerant actinomycetes isolated from the salt-affected soils is experimentally proved.

N<sub>2</sub>O, NO, N<sub>2</sub> and CO<sub>2</sub> emissions from tropical savanna and grassland of northern Australia: an incubation experiment with intact soil cores

Abstract. Strong seasonal variability of hygric and thermal soil conditions are a defining environmental feature in northern Australia. However, how such changes affect the soil–atmosphere exchange of nitrous oxide (N2O), nitric oxide (NO) and dinitrogen (N2) is still not well explored. By incubating intact soil cores from four sites (three savanna, one pasture) under controlled soil temperatures (ST) and soil moisture (SM) we investigated the release of the trace gas fluxes of N2O, NO and carbon dioxide (CO2). Furthermore, the release of N2 due to denitrification was measured using the helium gas flow soil core technique. Under dry pre-incubation conditions NO and N2O emissions were very low (<7.0 ± 5.0 μg NO-N m−2 h−1; <0.0 ± 1.4 μg N2O-N m−2 h−1) or in the case of N2O, even a net soil uptake was observed. Substantial NO (max: 306.5 μg N m−2 h−1) and relatively small N2O pulse emissions (max: 5.8 ± 5.0 μg N m−2 h−1) were recorded following soil wetting, but these pulses were short lived, lasting only up to 3 days. The total atmospheric loss of nitrogen was generally dominated by N2 emissions (82.4–99.3% of total N lost), although NO emissions contributed almost 43.2% to the total atmospheric nitrogen loss at 50% SM and 30 °C ST incubation settings (the contribution of N2 at these soil conditions was only 53.2%). N2O emissions were systematically higher for 3 of 12 sample locations, which indicates substantial spatial variability at site level, but on average soils acted as weak N2O sources or even sinks. By using a conservative upscale approach we estimate total annual emissions from savanna soils to average 0.12 kg N ha−1 yr−1 (N2O), 0.68 kg N ha−1 yr−1 (NO) and 6.65 kg N ha−1 yr−1 (N2). The analysis of long-term SM and ST records makes it clear that extreme soil saturation that can lead to high N2O and N2 emissions only occurs a few days per year and thus has little impact on the annual total. The potential contribution of nitrogen released due to pulse events compared to the total annual emissions was found to be of importance for NO emissions (contribution to total: 5–22%), but not for N2O emissions. Our results indicate that the total gaseous release of nitrogen from these soils is low and clearly dominated by loss in the form of inert nitrogen. Effects of seasonally varying soil temperature and moisture were detected, but were found to be low due to the small amounts of available nitrogen in the soils (total nitrogen <0.1%).

Filamentous actinobacteria of the saline soils of arid territories

A large number of actinomycetes (hundreds, thousands, and millions of CFU/g of soil) were isolated from saline soils in the territory of the landscapes of the Ber hillocks. A significantly smaller quantity of actinomycetes was isolated from the soda saline soils and sor saline soils that formed at the bottom of the ephemeral salty lakes in Buryatiya and in the estuary of the Syr-Darya River. Actinomycetes were represented in the studied soils by the Streptomyces, Micromonospora, Actinomadura, and Nocardiopsis genera. Among streptomycetes, the species of the Albus section and the Albus series predominated. The activity of the consumption of substrates by cultures of actinomycetes was largely influenced by conditions of preincubation. Obviously, the influence was related to the alteration in the metabolism of actinomycetes as one of the mechanisms of adaptation to the increased osmolarity of environment. The alcalotolerance, thermotolerance, and xerotolerance of halotolerant actinomycetes of the saline soils of arid territories were experimentally revealed.

Age‐related loss of neurotrophic signaling at the diaphragm muscle (871.4)

The age‐related mechanisms of diaphragm muscle (DIAm) sarcopenia are largely unknown. We hypothesize that sarcopenia may be related to neurotrophic signaling, specifically that of brain derived neurotrophic factor (BDNF) acting through the high‐affinity tropomyosin‐related kinase receptor subtype B (TrkB). In total 47, adult male TrkBF616A mice were examined at 6, 18, and 24 months of age (mo), representing survival rates of 100, 90, and 75%. At each age, ex vivo contractility of the DIAm was assessed to determine maximal force as well DIAm‐phrenic nerve neuromuscular transmission failure under the following preincubation conditions; control (vehicle) solution, BDNF, 1NMPP1 (inhibiting TrkB kinase activity), or co‐treatment of BDNF and 1NMPP1. Specific DIAm maximal twitch and isometric tetanic force, in addition to fatigue index were all negatively impacted at 24 mo (P蠄0.02). A significant interaction between age and neurotrophic treatment was found for DIAm‐phrenic nerve neuromuscular transmission failure (P蠄0.01). Specifically, at 6 and 18 mo BDNF decreased failure, while this was lost at 24 mo. Conversely, TrkB kinase inhibition increased failure at 6 mo, but this relationship was lost at both 18 and 24 mo. Taken together, neurotrophic signaling is key to the physiological function of the DIAm, however there is likely an age‐related loss of BDNF and TrkB signaling that detrimentally affects the DIAm.Grant Funding Source: Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

Nitrate regulates rhizobial and mycorrhizal symbiosis in common bean (Phaseolus vulgaris L.)

Nitrogen-limited conditions are considered to be a prerequisite for legume-rhizobial symbiosis, but the effects of nitrate-rich conditions on symbiotic status remain poorly understood. We addressed this issue by examining rhizobial (Rhizobim tropici) and arbusclar mycorrhizal (Glomus intraradices) symbiosis in Phaseolus vulgaris L. cv. Negro Jamapa under nitrate pre-incubation and continuous nitrate conditions. Our results indicate that nitrate pre-incubation, independent of the concentration, did not affect nodule development. However, the continuous supply of nitrate at high concentrations impaired nodule maturation and nodule numbers. Low nitrate conditions, in addition to positively regulating nodule number, biomass, and nitrogenase activity, also extended the span of nitrogen-fixing activity. By contrast, for arbuscular mycorrhizae, continuous 10 and 50 mmol/L nitrate increased the percent root length colonization, concomitantly reduced arbuscule size, and enhanced ammonia transport without affecting phosphate transport. Therefore, in this manuscript, we have proposed the importance of nitrate as a positive regulator in promoting both rhizobial and mycorrhizal symbiosis in the common bean.

Effect of Preincubation of Blood Culture Bottles in a BacT/Alert Unit Outside Laboratory Operating Hours on Detection Time

Background: The pre-storage condition of blood culture bottles prior to entering the automated blood culture system may affect the time to detection (TTD) of microorganisms and the final report days. Methods: We compared the TTD and final report days according to the pre-incubation conditions after ...