Transformation of Acinetobacter sp. Strain BD413 by Transgenic Sugar Beet DNA

The ability of Acinetobacter sp. strain BD413(pFG4ΔnptII) to take up and integrate transgenic plant DNA based on homologous recombination was studied under optimized laboratory conditions. Restoration of nptII, resulting in kanamycin-resistant transformants, was observed with plasmid DNA, plant DNA, and homogenates carrying the gene nptII. Molecular analysis showed that some transformants not only restored the 317-bp deletion but also obtained additional DNA.     

Anodal tDCS over the Primary Motor Cortex Facilitates Long-Term Memory Formation Reflecting Use-Dependent Plasticity

Previous research suggests that anodal transcranial direct current stimulation (tDCS) over the primary motor cortex (M1) modulates NMDA receptor dependent processes that mediate synaptic plasticity. Here we test this proposal by applying anodal versus sham tDCS while subjects practiced to flex the thumb as fast as possible (ballistic movements). Repetitive practice of this task has been shown to result in performance improvements that reflect use-dependent plasticity resulting from NMDA receptor mediated, long-term potentiation (LTP)-like processes. Using a double-blind within-subject cross-over design, subjects (n=14) participated either in an anodal or a sham tDCS session which were at least 3 months apart. Sham or anodal tDCS (1 mA) was applied for 20 min during motor practice and retention was tested 30 min, 24 hours and one week later. All subjects improved performance during each of the two sessions (p < 0.001) and learning gains were similar. Our main result is that long term retention performance (i.e. 1 week after practice) was significantly better when practice was performed with anodal tDCS than with sham tDCS (p < 0.001). This effect was large (Cohen’s d=1.01) and all but one subject followed the group trend. Our data strongly suggest that anodal tDCS facilitates long-term memory formation reflecting use-dependent plasticity. Our results support the notion that anodal tDCS facilitates synaptic plasticity mediated by an LTP-like mechanism, which is in accordance with previous research.     

DYn-2 Based Identification of Arabidopsis Sulfenomes

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H₂O₂) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H₂O₂ dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H₂O₂ treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.Among the different amino acids, the sulfur containing amino acids like cysteine are particularly susceptible to oxidation by reactive oxygen species (ROS)¹ (1, 2). Recent studies suggest that the sulfenome, the initial oxidation products of cysteine residues, functions as an intermediate state of redox signaling (3 –5). Thus, identifying the sulfenome under oxidative stress is a way to detect potential redox sensors (6, 7).This central role of the sulfenome in redox signaling provoked chemical biologists to develop strategies for sensitive detection and identification of sulfenylated proteins. The in situ trapping of the sulfenome is challenging because of two major factors: (1) the highly reactive, transient nature of sulfenic acids, which might be over-oxidized in excess of ROS, unless immediately protected by disulfide formation (7); (2) the intracellular compartmentalization of the redox state that might be disrupted during extraction procedures, resulting in artificial non-native protein oxidations (8, 9). Having a sulfur oxidation state of zero, sulfenic acids can react as both electrophile and nucleophile, however, direct detection methods are based on the electrophilic character of sulfenic acid (10). In 1974, Allison and coworkers reported a condensation reaction between the electrophilic sulfenic acid and the nucleophile dimedone (5,5-dimethyl-1,3-cyclohexanedione), producing a corresponding thioether derivative (11). This chemistry is highly selective and, since then, has been exploited to detect dimedone modified sulfenic acids using mass spectrometry (12). However, dimedone has limited applications for cellular sulfenome identification because of the lack of a functional group to enrich the dimedone tagged sulfenic acids. Later, dimedone-biotin/fluorophores conjugates have been developed, which allowed sensitive detection and enrichment of sulfenic acid modified proteins (13 –15). This approach, however, was not always compatible with in vivo cellular sulfenome analysis, because the biotin/fluorophores-conjugated dimedone is membrane impermeable (9) and endogenous biotinylated proteins might appear as false positives.More recently, the Carroll lab has developed DYn-2, a sulfenic acid specific chemical probe. This chemical probe consists of two functional units: a dimedone scaffold for sulfenic acid recognition and an alkyne chemical handle for enrichment of labeled proteins (9). Once the sulfenic acids are tagged with the DYn-2 probe, they can be biotinylated through click chemistry (16). The click reaction used here is a copper (I)-catalyzed azide-alkyne cycloaddition reaction (17), also known as azide-alkyne Huisgen cycloaddition (16). With this chemistry, a complex is formed between the alkyne functionalized DYn-2 and the azide functionalized biotin. This biotin functional group facilitates downstream detection, enrichment, and mass spectrometry based identification (Fig. 1). In an evaluation experiment, DYn-2 was found to efficiently detect H₂O₂-dependent sulfenic acid modifications in recombinant glutathione peroxidase 3 (Gpx3) of budding yeast (18). Moreover, it was reported that DYn-2 is membrane permeable, non-toxic, and a non-influencer of the intracellular redox balance (17, 18). Therefore, DYn-2 has been suggested as a global sulfenome reader in living cells (17, 18), and has been applied to investigate epidermal growth factor (EGF) mediated protein sulfenylation in a human epidermoid carcinoma A431 cell line and to identify intracellular protein targets of H₂O₂ during cell signaling (17).Open in a separate windowFig. 1.Schematic views of the molecular mechanism of the DYn-2 probe and the strategy to identify DYn-2 trapped sulfenylated proteins. A, DYn-2 specifically detects sulfenic acid modifications, but no other thiol modifications. B, Biotinylation of the DYn-2 tagged proteins by click reaction. C, Once DYn-2 tagged proteins are biotinylated, a streptavidin-HRP (Strep-HRP) blot visualizes sulfenylation, or alternatively, after enrichment on avidin beads, proteins are identified by mass spectrometry analysis.Here, we selected the DYn-2 probe to identify the sulfenome in plant cells under oxidative stress. Through a combination of biochemical, immunoblot and mass spectrometry techniques, and TAIR10 database and SUBA3-software predictions, we can claim that DYn-2 is able to detect sulfenic acids on proteins located in different subcellular compartments of plant cells. We identified 226 sulfenylated proteins in response to an H₂O₂ treatment of Arabidopsis cell suspensions, of which 123 proteins are new candidates for cysteine oxidative post-translational modification (PTM) events.     

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.     

Traditional and Emerging Lifestyle Risk Behaviors and All-Cause Mortality in Middle-Aged and Older Adults: Evidence from a Large Population-Based Australian Cohort

Background

Lifestyle risk behaviors are responsible for a large proportion of disease burden worldwide. Behavioral risk factors, such as smoking, poor diet, and physical inactivity, tend to cluster within populations and may have synergistic effects on health. As evidence continues to accumulate on emerging lifestyle risk factors, such as prolonged sitting and unhealthy sleep patterns, incorporating these new risk factors will provide clinically relevant information on combinations of lifestyle risk factors.

Methods and Findings

Using data from a large Australian cohort of middle-aged and older adults, this is the first study to our knowledge to examine a lifestyle risk index incorporating sedentary behavior and sleep in relation to all-cause mortality. Baseline data (February 2006– April 2009) were linked to mortality registration data until June 15, 2014. Smoking, high alcohol intake, poor diet, physical inactivity, prolonged sitting, and unhealthy (short/long) sleep duration were measured by questionnaires and summed into an index score. Cox proportional hazards analysis was used with the index score and each unique risk combination as exposure variables, adjusted for socio-demographic characteristics.During 6 y of follow-up of 231,048 participants for 1,409,591 person-years, 15,635 deaths were registered. Of all participants, 31.2%, 36.9%, 21.4%, and 10.6% reported 0, 1, 2, and 3+ risk factors, respectively. There was a strong relationship between the lifestyle risk index score and all-cause mortality. The index score had good predictive validity (c index = 0.763), and the partial population attributable risk was 31.3%. Out of all 96 possible risk combinations, the 30 most commonly occurring combinations accounted for more than 90% of the participants. Among those, combinations involving physical inactivity, prolonged sitting, and/or long sleep duration and combinations involving smoking and high alcohol intake had the strongest associations with all-cause mortality. Limitations of the study include self-reported and under-specified measures, dichotomized risk scores, lack of long-term patterns of lifestyle behaviors, and lack of cause-specific mortality data.

Conclusions

Adherence to healthy lifestyle behaviors could reduce the risk for death from all causes. Specific combinations of lifestyle risk behaviors may be more harmful than others, suggesting synergistic relationships among risk factors.     

Inhibition of <Emphasis Type="Italic">Candida</Emphasis> <Emphasis Type="Italic">albicans</Emphasis> Biofilm Formation by Antimycotics Released from Modified Polydimethyl Siloxane

Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.