Effects of salinity on microbial tolerance to drying and rewetting

Soil salinity and fluctuations in soil matric potential are stressors for soil microorganisms which, in turn, may affect soil organic matter turnover. In response to salinity and low soil water content, many microorganisms accumulate osmolytes. Therefore, it is conceivable that microorganisms in saline soils are more tolerant to drying and rewetting (DRW) stress than those in non-saline soils. An experiment was carried out with three different salinity levels: electrical conductivity (EC_1:5) 0, 2 and 4 dS m^?1 (EC0, EC2, EC4), and two water treatments: a constantly moist control or two DRW cycles. Respiration as an indicator of microbial activity was measured throughout the 59 days of incubation. At the end of the second dry period (day 35) and at the end of the following moist incubation (day 59), microbial biomass and microbial community structure were determined by phospholipid fatty acid (PLFA) analysis. Increasing salinity decreased microbial activity but did not affect its resistance to DRW. On day 59, cumulative respiration decreased in the order EC0 > EC2 > EC4 with no differences between water treatments. Fungal biomass was negatively affected by salinity at the end of the experiment, while bacterial biomass was unaffected. Microbial community structure in moist treatments differed between salinity levels, with EC4 influencing microbial community structure earlier than EC2. The resistance of microbial communities to DRW stress was salt level dependent; only beyond a critical salinity level adaptation to salt stress was able to reduce the impact of water stress on microbial community structure.     

Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes

Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid-protein nanodomains, termed "rafts". In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids ("raft lipids", e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena.     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Seeing 'where' through the ears: effects of learning-by-doing and long-term sensory deprivation on localization based on image-to-sound substitution

Background

Sensory substitution devices for the blind translate inaccessible visual information into a format that intact sensory pathways can process. We here tested image-to-sound conversion-based localization of visual stimuli (LEDs and objects) in 13 blindfolded participants.

Methods and Findings

Subjects were assigned to different roles as a function of two variables: visual deprivation (blindfolded continuously (Bc) for 24 hours per day for 21 days; blindfolded for the tests only (Bt)) and system use (system not used (Sn); system used for tests only (St); system used continuously for 21 days (Sc)). The effect of learning-by-doing was assessed by comparing the performance of eight subjects (BtSt) who only used the mobile substitution device for the tests, to that of three subjects who, in addition, practiced with it for four hours daily in their normal life (BtSc and BcSc); two subjects who did not use the device at all (BtSn and BcSn) allowed assessment of its use in the tasks we employed. The impact of long-term sensory deprivation was investigated by blindfolding three of those participants throughout the three week-long experiment (BcSn, BcSn/c, and BcSc); the other ten subjects were only blindfolded during the tests (BtSn, BtSc, and the eight BtSt subjects). Expectedly, the two subjects who never used the substitution device, while fast in finding the targets, had chance accuracy, whereas subjects who used the device were markedly slower, but showed much better accuracy which improved significantly across our four testing sessions. The three subjects who freely used the device daily as well as during tests were faster and more accurate than those who used it during tests only; however, long-term blindfolding did not notably influence performance.

Conclusions

Together, the results demonstrate that the device allowed blindfolded subjects to increasingly know where something was by listening, and indicate that practice in naturalistic conditions effectively improved “visual” localization performance.     

Estimation of the quantity of bacteria encapsulated in Lentikats Biocatalyst via phospholipid fatty acids content: a preliminary study

The content of phospholipid fatty acids (PLFA) was determined in samples of polyvinyl alcohol lenses (Lentikats Biocatalyst, LB) with encapsulated Paracoccus denitrificans withdrawn during long-term denitrification experiments. The total PLFA content correlated highly with specific denitrification activities of LB as well as biomass estimation based on image analyses of microscopic photos. The results confirmed the applicability of PLFA determination for estimation of the amount of living encapsulated microbial biomass during biotechnological applications.     

Expression of human aldo-keto reductase 1C2 in cell lines of peritoneal endometriosis: potential implications in metabolism of progesterone and dydrogesterone and inhibition by progestins

The human aldo-keto reductase AKR1C2 converts 5α-dihydrotestosterone to the less active 3α-androstanediol and has a minor 20-ketosteroid reductase activity that metabolises progesterone to 20α-hydroxyprogesterone. AKR1C2 is expressed in different peripheral tissues, but its role in uterine diseases like endometriosis has not been studied in detail. Some progestins used for treatment of endometriosis inhibit AKR1C1 and AKR1C3, with unknown effects on AKR1C2. In this study we investigated expression of AKR1C2 in the model cell lines of peritoneal endometriosis, and examined the ability of recombinant AKR1C2 to metabolise progesterone and progestin dydrogesterone, as well as its potential inhibition by progestins. AKR1C2 is expressed in epithelial and stromal endometriotic cell lines at the mRNA level. The recombinant enzyme catalyses reduction of progesterone to 20α-hydroxyprogesterone with a 10-fold lower catalytic efficiency than the major 20-ketosteroid reductase, AKR1C1. AKR1C2 also metabolises progestin dydrogesterone to its 20α-dihydrodydrogesterone, with 8.6-fold higher catalytic efficiency than 5α-dihydrotestosterone. Among the progestins that are currently used for treatment of endometriosis, dydrogesterone, medroxyprogesterone acetate and 20α-dihydrodydrogesterone act as AKR1C2 inhibitors with low μM K(i) values in vitro. Their potential in vivo effects should be further studied.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K_cat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5‐fold increased specific activity (15°C) and over 100 times prolonged half‐life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity‐altering amino acid substitutions were from non‐charged to non‐charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc.     

Treatment-independent miRNA signature in blood of wilms tumor patients

ABSTRACT: BACKGROUND: Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared themiRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOPprotocol 2001. RESULTS: We did not find a significant difference between miRNA signature of both groups. However both, Wilmstumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. Thesignature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5% and of patients afterchemotherapy an accuracy of 97.0%, each as compared to healthy controls. CONCLUSION: Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of fourweeks preoperative chemotherapy treatment.