Physiology of solvent tolerance in Pseudomonas putida S12

Hydrophobic organic solvents, like toluene, are toxic for living organisms. This toxicity is an important drawback in the environmental biotechnology as well as in the application of solvents in the production of fine chemicals by whole-cell biotransformations. The effects of organic solvents on micro-organisms have been studied extensively. It was shown that the toxicity of hydrophobic organic solvents is mainly caused by the ability of such solvents to intercalate and accumulate in biological membranes.In the last decade, however, several strains that can survive the presence of toxic organic solvents have been isolated. One of the solvent-tolerant strains is Pseudomonas putida S12, studied in this thesis. This strain can grow in the presence of a second phase of organic solvents that have a log P O/W (logarithm of the partition coefficient between octanol and water) value equal to or higher than 2.3. P. putida S12 is able to survive the presence of solvents because of different adaptation mechanisms. This strain can suppress the effect of organic solvents on the membranes by a cis to trans isomerization of the unsaturated fatty acids of the membrane.A further adaptation mechanism is presented in this thesis. It is shown that cells adapted to toluene posses an active export system for toluene. Therefore, P. putida S12 compensates not only the toxic effects of organic solvents on the membrane, but decrease also actively the amount of the toxic solvent in the cell. In the presence of the efflux system the concentration of a solvent in the bacterial membrane can be below the theoretical equilibrium. This active efflux system depends on the proton motive force.Since we reported the presence of an active export system for solvents, it has been suggested repeatedly, that this system is connected with the well-described efflux systems for antibiotics. Indeed, the adaptation of P. putida S12 to toluene enhances the resistance of this strain to various chemically and structurally unrelated antibiotics, with different targets in the cell.However, we could demonstrate that efflux system for toluene in P. putida S12 does not export antibiotics. This efflux system is specific for solvents like toluene and p -xylene. Therefore, the adaptation to an organic solvent must activate other mechanisms responsible for the resistance towards these antibiotics. Likely, this is connected to a general stress response. This general stress response may also cause the decrease of the cell-envelope permeability discussed in the thesis.The broad effect solvents have even on the solvent-tolerant strain P. putida S12 are demonstrated. The presence of toluene reduces the maximum growth yield and increases the maintenance requirement. Interestingly, other solvents had a similar effect as toluene as long as they reached the same concentration in the bacterial membrane. Not the chemical structure but the amount of solvent accumulated in the bacterial membrane determines the effect of a solvent on the cells. Therefore, results obtained with toluene can be extrapolated to other solvents as well.</p

Circulating vaspin is unrelated to insulin sensitivity in a cohort of nondiabetic humans

Objective: To study the association of vaspin with glucose metabolism. Design: Cross-sectional and intervention study. Subjects and methods: The association of serum vaspin with metabolic and anthropometric characteristics was investigated in 108 volunteers. Euglycemic–hyperinsulinemic clamps (EHC) were performed in 83 of the participants. Changes of circulating vaspin levels were additionally studied in a crossover study using 300 min EHC with lipid versus saline infusion (n=10). Results: Neither glucose tolerance status nor insulin sensitivity, both as measured using EHCs and using homeostasis model assessment for insulin resistance (HOMA-IR), was significantly associated with serum vaspin in the cross-sectional study. Furthermore, there was no effect of short-term lipid-induced insulin resistance due to a 300 min intravenous lipid challenge on circulating vaspin. However, circulating vaspin levels were significantly elevated in women using oral contraceptives (OC), both compared to women without OC intake (1.17±0.26 vs 0.52±0.09 ng/ml, P=0.02) and males (1.17±0.26 vs 0.29±0.04 ng/ml, P=0.01). After exclusion of OC using females and stratification according to body mass index (BMI), a significant sexual dimorphism in subjects with a BMI <25 kg/m2 was observed (males 0.21±0.04 ng/ml versus females 0.70±0.16 ng/ml, P=0.009). Conclusion: Our results support the existence of a sexual dimorphism regarding circulating vaspin. The lack of an association of serum vaspin with HOMA-IR and M value indicates, however, no major role for vaspin concerning insulin sensitivity in nondiabetic humans

Metabolic effects of diets differing in glycaemic index depend on age and endogenous GIP

Aims/hypothesis High- vs low-glycaemic index (GI) diets unfavourably affect body fat mass and metabolic markers in rodents. Different effects of these diets could be age-dependent, as well as mediated, in part, by carbohydrate-induced stimulation of glucose-dependent insulinotrophic polypeptide (GIP) signalling. Methods Young-adult (16 weeks) and aged (44 weeks) male wild-type (C57BL/6J) and GIP-receptor knockout (Gipr −/− ) mice were exposed to otherwise identical high-carbohydrate diets differing only in GI (20–26 weeks of intervention, n = 8–10 per group). Diet-induced changes in body fat distribution, liver fat, locomotor activity, markers of insulin sensitivity and substrate oxidation were investigated, as well as changes in the gene expression of anorexigenic and orexigenic hypothalamic factors related to food intake. Results Body weight significantly increased in young-adult high- vs low-GI fed mice (two-way ANOVA, p < 0.001), regardless of the Gipr genotype. The high-GI diet in young-adult mice also led to significantly increased fat mass and changes in metabolic markers that indicate reduced insulin sensitivity. Even though body fat mass also slightly increased in high- vs low-GI fed aged wild-type mice (p < 0.05), there were no significant changes in body weight and estimated insulin sensitivity in these animals. However, aged Gipr −/− vs wild-type mice on high-GI diet showed significantly lower cumulative net energy intake, increased locomotor activity and improved markers of insulin sensitivity. Conclusions/interpretation The metabolic benefits of a low-GI diet appear to be more pronounced in younger animals, regardless of the Gipr genotype. Inactivation of GIP signalling in aged animals on a high-GI diet, however, could be beneficial

Drainage of a deep magma reservoir near Mayotte inferred from seismicity and deformation

The dynamics of magma deep in the Earth’s crust are difficult to capture by geophysical monitoring. Since May 2018, a seismically quiet area offshore of Mayotte in the western Indian Ocean has been affected by complex seismic activity, including long-duration, very-long-period signals detected globally. Global Navigation Satellite System stations on Mayotte have also recorded a large surface deflation offshore. Here we analyse regional and global seismic and deformation data to provide a one-year-long detailed picture of a deep, rare magmatic process. We identify about 7,000 volcano-tectonic earthquakes and 407 very-long-period seismic signals. Early earthquakes migrated upward in response to a magmatic dyke propagating from Moho depth to the surface, whereas later events marked the progressive failure of the roof of a magma reservoir, triggering its resonance. An analysis of the very-long-period seismicity and deformation suggests that at least 1.3 km3 of magma drained from a reservoir of 10 to 15 km diameter at 25 to 35 km depth. We demonstrate that such deep offshore magmatic activity can be captured without any on-site monitoring

Solving integral equations in η→3π\eta\to 3\pi

A dispersive analysis of $\eta\to 3\pi$ decays has been performed in the past by many authors. The numerical analysis of the pertinent integral equations is hampered by two technical difficulties: i) The angular averages of the amplitudes need to be performed along a complicated path in the complex plane. ii) The averaged amplitudes develop singularities along the path of integration in the dispersive representation of the full amplitudes. It is a delicate affair to handle these singularities properly, and independent checks of the obtained solutions are demanding and time consuming. In the present article, we propose a solution method that avoids these difficulties. It is based on a simple deformation of the path of integration in the dispersive representation (not in the angular average). Numerical solutions are then obtained rather straightforwardly. We expect that the method also works for $\omega\to 3\pi$.Comment: 11 pages, 10 Figures. Version accepted for publication in EPJC. The ancillary files contain an updated set of fundamental solutions. The numerical differences to the former set are tiny, see the READMEv2 file for detail

The 3′-Terminal Hexamer Sequence of Classical swine fever virus RNA Plays a Role in Negatively Regulating the IRES-Mediated Translation

The 3′ untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3′ UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5′ and 3′ UTRs, we found that the 3′ UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3′-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3′-ends such as the poly(A) or the 3′ UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3′ UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5′ UTR. In conclusion, we have found that the 3′-end terminal sequence can play a role in regulating the translation of CSFV

Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes

Production of HIV Particles Is Regulated by Altering Sub-Cellular Localization and Dynamics of Rev Induced by Double-Strand RNA Binding Protein

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1–dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication