Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents

Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.     

Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm,Helicoverpa armigera (Hubner)

American bollworm (ABW), Helicoverpa armigera (Hubner), is considered as a major pest of cotton, Gossypium hirsutum, all over the globe. Due to its destructive feeding nature and continuous consumption of the same chemicals, it devolved resistant against many insecticides. Therefore, a combined application of bio- and synthetic-pesticide need to evaluate against this pest. The entomopathogenic viruses like nuclear polyhedrosis virus (NPV), a member of baculoviruses, can be the potential candidates for better control against ABW. The present study was conducted to assess the comparative efficacy of NPV and Spinosad 240SC (with the concentration of 250 mL · ha￣?) against ABW in the controlled environment. The ABW was treated with different concentrations of NPV and Spinosad separately and in a combination of NPV with 0.1% Spinosad. The results revealed that highest concentrations showed highest mortality (95%) followed by 95%, 92%, 84%, 82% and 78% mortality at 1 × 10⁹, 1 × 10⁸, 1 × 10⁷, 1 × 10⁶ and 1 × 10⁵ POBs, respectively. Spinosad when mixed in diet give 100% mortality at 0.8% followed by 50.87%, 42.10%, 29.82%, 26.31% and 22.80% mortality at 0.4%, 0.2%, 0.1%, 0.5% and 0.025% respectively. The results of this study revealed that microbial control of ABW through NPV is an effective tool. The repeated use of synthetic pesticides caused the resurgence of many insect pests, and this study results would provide useful insight to build a framework for future investigations for the management of many major insect pests.     

Novel oral transforming growth factor-β signaling inhibitor potently inhibits postsurgical adhesion band formation

Here, we have investigated the therapeutic potency of EW-7197, a transforming growth factor-β type I receptor kinase inhibitor, against postsurgical adhesion band formation. Our results showed that this pharmacological inhibitor prevented the frequency and the stability of adhesion bands in mice model. We have also shown that downregulation of proinflammatory cytokines, reduce submucosal edema, attenuation of proinflammatory cell infiltration, inhibition of oxidative stress, decrease in excessive collagen deposition, and suppression of profibrotic genes at the site of surgery are some of the mechanisms by which EW-7197 elicits its protective responses against adhesion band formation. These results clearly suggest that EW-7197 has novel therapeutic properties against postsurgical adhesion band formation with clinically translational potential of inhibiting key pathological responses of inflammation and fibrosis in postsurgery patients.     

Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO₂⁻) (30%), nitrous oxide (N₂O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [¹⁵N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N₂O with two molecular mass ions: one at 44 Da, corresponding to ¹⁴N¹⁴NO, and the second at 45 Da, corresponding to ¹⁵N¹⁴NO. The nonlabeled N₂O could be formed only from -NO₂, whereas the ¹⁵N-labeled one was presumed to originate from a nitramine group (¹⁵N-¹⁴NO₂) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C₂H₅N₃O₃. When the experiment was repeated with ring-labeled [¹⁵N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-¹⁴C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in ¹⁴CO₂ (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO₂ and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2

Cyclic nitramine explosives, RDX, HMX, and CL-20 are hydrophobic pollutants with very little aqueous solubility. In sediment and soil environments, they are often attached to solid surfaces and/or trapped in pores and distribute heterogeneously in aqueous environments. For efficient bioremediation of these explosives, the microorganism(s) must access them by chemotaxis ability. In the present study, we isolated an obligate anaerobic bacterium Clostridium sp. strain EDB2 from a marine sediment. Strain EDB2, motile with numerous peritrichous flagella, demonstrated chemotactic response towards RDX, HMX, CL-20, and NO(2)(-). The three explosives were biotransformed by strain EDB2 via N-denitration with concomitant release of NO(2)(-). Biotransformation rates of RDX, HMX, and CL-20 by the resting cells of strain EDB2 were 1.8+/-0.2, 1.1+/-0.1, and 2.6+/-0.2nmol h(-1)mgwet biomass(-1) (mean+/-SD; n=3), respectively. We found that commonly seen RDX metabolites such as TNX, methylenedinitramine, and 4-nitro-2,4-diazabutanal neither produced NO(2)(-) during reaction with strain EDB2 nor they elicited chemotaxis response in strain EDB2. The above data suggested that NO(2)(-) released from explosives during their biotransformation might have elicited chemotaxis response in the bacterium. Biodegradation and chemotactic ability of strain EDB2 renders it useful in accelerating the bioremediation of explosives under in situ conditions.     

Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia

Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.     

A predictive model for respiratory syncytial virus (RSV) hospitalisation of premature infants born at 33–35 weeks of gestational age,based on data from the Spanish FLIP study

Background

The aim of this study, conducted in Europe, was to develop a validated risk factor based model to predict RSV-related hospitalisation in premature infants born 33–35 weeks'' gestational age (GA).

Methods

The predictive model was developed using risk factors captured in the Spanish FLIP dataset, a case-control study of 183 premature infants born between 33–35 weeks'' GA who were hospitalised with RSV, and 371 age-matched controls. The model was validated internally by 100-fold bootstrapping. Discriminant function analysis was used to analyse combinations of risk factors to predict RSV hospitalisation. Successive models were chosen that had the highest probability for discriminating between hospitalised and non-hospitalised infants. Receiver operating characteristic (ROC) curves were plotted.

Results

An initial 15 variable model was produced with a discriminant function of 72% and an area under the ROC curve of 0.795. A step-wise reduction exercise, alongside recalculations of some variables, produced a final model consisting of 7 variables: birth ± 10 weeks of start of season, birth weight, breast feeding for ≤ 2 months, siblings ≥ 2 years, family members with atopy, family members with wheeze, and gender. The discrimination of this model was 71% and the area under the ROC curve was 0.791. At the 0.75 sensitivity intercept, the false positive fraction was 0.33. The 100-fold bootstrapping resulted in a mean discriminant function of 72% (standard deviation: 2.18) and a median area under the ROC curve of 0.785 (range: 0.768–0.790), indicating a good internal validation. The calculated NNT for intervention to treat all at risk patients with a 75% level of protection was 11.7 (95% confidence interval: 9.5–13.6).

Conclusion

A robust model based on seven risk factors was developed, which is able to predict which premature infants born between 33–35 weeks'' GA are at highest risk of hospitalisation from RSV. The model could be used to optimise prophylaxis with palivizumab across Europe.     

Systemic Induction of Photosynthesis via Illumination of the Shoot Apex Is Mediated Sequentially by Phytochrome B,Auxin and Hydrogen Peroxide in Tomato

Systemic signaling of upper leaves promotes the induction of photosynthesis in lower leaves, allowing more efficient use of light flecks. However, the nature of the systemic signals has remained elusive. Here, we show that preillumination of the tomato (Solanum lycopersicum) shoot apex alone can accelerate photosynthetic induction in distal leaves and that this process is light quality dependent, where red light promotes and far-red light delays photosynthetic induction. Grafting the wild-type rootstock with a phytochome B (phyB) mutant scion compromised light-induced photosynthetic induction as well as auxin biosynthesis in the shoot apex, auxin signaling, and RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1)-dependent hydrogen peroxide (H₂O₂) production in the systemic leaves. Light-induced systemic H₂O₂ production in the leaves of the rootstock also was absent in plants grafted with an auxin-resistant diageotropica (dgt) mutant scion. Cyclic electron flow around photosystem I and associated ATP production were increased in the systemic leaves by exposure of the apex to red light. This enhancement was compromised in the systemic leaves of the wild-type rootstock with phyB and dgt mutant scions and also in RBOH1-RNA interference leaves with the wild type as scion. Silencing of ORANGE RIPENING, which encodes NAD(P)H dehydrogenase, compromised the systemic induction of photosynthesis. Taken together, these results demonstrate that exposure to red light triggers phyB-mediated auxin synthesis in the apex, leading to H₂O₂ generation in systemic leaves. Enhanced H₂O₂ levels in turn activate cyclic electron flow and ATP production, leading to a faster induction of photosynthetic CO₂ assimilation in the systemic leaves, allowing plants better adaptation to the changing light environment.As a consequence of their sessile lifestyle, plants have evolved a high capacity for the regulation of physiology, growth, and development that facilitates survival in a constantly changing environment. Environmental stimuli perceived within an organ not only influence morphogenetic and physiological changes within that organ but also generate systemic effects in other organs that are remote from the site of signal perception. This crucial phenomenon is called systemic signaling or systemic regulation. Systemic signaling prepares other tissues of a plant for future challenges that may initially only be sensed by a few local tissues or cells. Several types of systemic responses are known. These include systemic acquired resistance, which is typically activated by pathogens such as viruses, bacteria, and fungi (Fu and Dong, 2013), induced systemic resistance, which is triggered by beneficial soil microorganisms or others (Pieterse and Dicke, 2007), and systemic acquired acclimation, which is initiated by abiotic stresses such as high light, UV radiation, heat, cold, and salinity (Mittler and Blumwald, 2015).The light utilization efficiency of photosynthesis is important for the survival of understory plants and plants growing in canopies. In particular, the efficient use of the energy contained in light (sun) flecks is important because light flecks contribute up to 60% to 80% of photosynthetically active radiation received by understory plants (Pearcy and Seemann, 1990; Leakey et al., 2003, 2005). Earlier studies have shown the existence of systemic regulation of stomatal development and of photosynthesis in developing leaves in response to environmental signals perceived by mature leaves, such as changing irradiance and atmospheric CO₂ conditions (Lake et al., 2002; Coupe et al., 2006; Araya et al., 2008). Phytochome B (phyB) is important in the transmission of the systemic signals that modulate stomatal development in young leaves of Arabidopsis (Arabidopsis thaliana; Casson and Hetherington, 2014). In tomato (Solanum lycopersicum), there are two forms of phyB, phyB1 and phyB2, that work together to mediate red (R) light-induced responses, such as hypocotyl elongation and greening in seedlings (Hauser et al., 1995; Weller et al., 2000).Photosynthesis is completely switched off in the dark, specifically to prevent futile cycling of metabolites through the reductive and oxidative pentose phosphate pathways. Hence, leaves need time to reactivate the enzymes of carbon assimilation after a period of darkness. The time taken to reach maximum net rates of photosynthesis upon illumination is called photosynthetic induction (Walker, 1973). Systemic signaling also has been observed for the regulation of photosynthesis in relation to leaf ontology in understory plants (Montgomery and Givnish, 2008). The uppermost leaves, which are generally the first to receive sunlight, display faster photosynthetic induction times than understory leaves (Bai et al., 2008). Photosynthetic induction in understory leaves is enhanced by the preillumination of upper leaves but not lower leaves, suggesting a directional signal transfer (Hou et al., 2015). While this process allows plants to use the light energy in sun flecks more efficiently, the nature of the systemic signals and their transmission pathways remain largely unresolved. Although systemic signaling between different leaf ranks has been suggested to occur through the xylem (Thorpe et al., 2007) and also via electrical signals (Zimmermann et al., 2009), it is likely that systemic signals also pass through the phloem (Turgeon and Wolf, 2009; Hou et al., 2015). In addition, the phytohormone auxin is produced in the shoot apex and redistributed throughout the shoot by rapid nonpolar phloem transport (Ljung et al., 2001). Changes in the light environment can dramatically alter auxin homeostasis, which is regulated in a light quality- and photoreceptor-dependent manner (Halliday et al., 2009).The photosynthetic electron transport chain exhibits enormous flexibility in the relative rates of NADPH and ATP production in order to accommodate the varying requirements of metabolism (Foyer et al., 2012). Noncyclic, pseudocyclic, and cyclic electron flow (CEF) pathways operate in the photosynthetic electron transport chain to drive the proton gradient across the thylakoid membrane (Allen, 2003). Photosynthetic induction is not only associated with the activation of the light- and thiol-dependent activation of carbon assimilation enzymes but also dependent on a high rate of CEF to drive ATP synthesis (Foyer et al., 1992). Considerable overreduction of the electron transport acceptors occurs during the photosynthetic induction period, and this continues until carbon assimilation can be activated. CEF around PSI, an essential component of photosynthesis, drives the proton gradient in a situation when NADP reduction has reached its highest capacity and this essential electron acceptor is no longer available (Yamori et al., 2015; Yamori and Shikanai, 2016). CEF is particularly sensitive to the reduction-oxidation (redox) status of the chloroplast, which in turn is responsive to cellular redox homeostasis. Oxidants such as hydrogen peroxide (H₂O₂), which are produced by pseudocyclic electron flow in the chloroplasts, play a crucial role in the activation of CEF through modulation of the activity of the NADPH-plastoquinone reductase complex (Strand et al., 2015). Hormone-mediated generation of H₂O₂ also can stimulate CO₂ assimilation (Jiang et al., 2012).Auxins such as indole-3-acetic acid (IAA) generate H₂O₂ (Ivanchenko et al., 2013; Peer et al., 2013) and can regulate CO₂ assimilation (Bidwell and Turner, 1966; Hayat et al., 2009; Peng et al., 2013). Therefore, we used tomato plants to test the hypothesis that the systemic signaling that regulates photosynthetic induction in understory leaves arises from light-induced changes in auxin and H₂O₂ homeostasis involving the modulation of CEF in systemic leaves. We present evidence showing that R light perceived in the shoot apex by a phyB-dependent pathway alters IAA signaling in a systemic manner. IAA signals from the apex, perceived in distal leaves, trigger systemic H₂O₂ production that accelerates photosynthetic induction by increasing CEF-dependent ATP production in the systemic leaves. These findings provide new insights into the elaborate plant regulatory network that allows light adaptation in different organs.     

Metabolite production during transformation of 2,4,6-trinitrotoluene (TNT) by a mixed culture acclimated and maintained on crude oil-containing media

Metabolites formed during 2,4,6-trinitrotoluene (TNT) removal by a mixed bacterial culture (acclimated and maintained on crude oil-containing medium and capable of high rates of TNT removal) were characterized. In resting cell experiments in the absence of glucose, 46.2 mg/l TNT were removed in 171 h (87.5% removal), with a combined total formation of 7.7 mg/l amino-4,6-dinitrotoluene (ADNT) and 0.3 mg/l 4,4-azoxytetranitrotoluene and 2,4-azoxytetranitrotoluene, leaving 70% of the initial TNT unaccounted for. In the presence of glucose, resting cells removed 45.4 mg/l TNT in 49 h (95.5% removal), with 9.1 mg/l ADNT and 2.4 mg/l azoxy compounds being produced, leaving 70.3% of the TNT unaccounted for. Growing cells (glucose present) were capable of removing 44.2 mg/l TNT within 21 h (97.9% removal), with the concomitant formation of 1.8 mg/l ADNTs and 2.2 mg/l azoxy compounds. Denitrated TNT in the form of 2,6-dinitrotoluene was also produced in growing cells with a maximum amount of 1.31 mg/l after 28 h, followed by a slight decrease with time, leaving 88.5% of the initial TNT unaccounted for after 171 h. Radiolabeled ¹⁴C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.     

Fluorinated aminoglycosides and their mechanistic implication for aminoglycoside 3'-phosphotransferases from Gram-negative bacteria

Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.