Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.Key words: DNA methylation, upper aerodigestive tract, cancer, risk factors, biomarkers     

d-Xylose detection in Escherichia coli by a xylose binding protein-dependent response

A gene circuit for the controlled expression of a marker gene and for the assay of xylose concentration in Escherichia coli has been designed and tested. The xylF coding sequence for the xylose binding protein (XBP) was cloned in pT7T318U downstream from the promoter for xylanase A from B. subtilis (Pbsu), together with the GFP coding sequence (gfp) under the control of the xylF promoter, forming the pT7T3-GFP-XBP construct. GFP fluorescence in Escherichia coli JW3538-1 xylF—transformed with pT7T3-GFP-XBP was approximately 1.4× higher after 520 min growth in the presence of 5 mM xylose than in cells transformed with pT7T3-GFP. Under saturating xylose concentration, flow cytometry analysis showed that all cells resulted in homogeneous populations, and the population with XBP showed a fluorescence greater than that without XBP. Activity of the xylF promoter in cells transformed with pT7T3-GFP-XBP was ∼40% higher than with the pT7T3-GFP. No response was observed with arabinose and ribose, showing that the expression effects were specific for xylose, demonstrating the potential use of the gene circuit as a biosensor.     

Antimicrobial susceptibility and sessile behaviour of bacteria isolated from a minimally processed vegetables plant

Abstract

In this study, 20 heterotrophic bacteria from a minimally processed vegetables (MPV) plant were tested for their susceptibilities to five antibiotics (tetracycline, erythromycin, ampicillin, levofloxacin and ciprofloxacin), their (co)aggregation abilities and their survival under gastric simulated conditions. Peracetic acid (PA) and sodium hypochlorite (SH), both at 50?ppm, were evaluated for their abilities to control biofilms of these bacteria. In general, the Gram-negative bacteria were found to be more resistant to the selected antibiotics. Two isolates, Rhanella aquatilis and Stenotrophomonas maltophilia, demonstrated multidrug resistance. Only Rhodococcus erythropolis presented aggregation potential, while no bacterium survived under the gastric conditions. The biofilm experiments showed PA as less efficient than SH in killing biofilms and neither of the disinfectants was able to fully eliminate the biofilms. Significant regrowth was observed for most of the biofilms. The results indicate that alternative and/or complementary disinfection strategies are required to guarantee food safety.     

Pseudomonas putida are environmental reservoirs of antimicrobial resistance to β-lactamic antibiotics

The adaptive flexibility of bacteria largely contributes to the emergence of antibiotic resistance, eventually leading to the predictable failure of current antimicrobial therapies. It is of utmost importance to improve current approaches and implement new ways to control bacterial growth and proliferation. A promising strategy lies in unraveling the antimicrobial resistance (AMR) dynamics in environmental reservoirs, namely in soil. Environmental microorganisms are antibiotic producers and generally also carriers of AMR mechanisms. Therefore, soil samples were collected from areas distinctly influenced by men: rural farms and urban fluvial shores. Globally, microbial communities collected in farms revealed the highest antibiotic resistance potential. Largely predominant Gram-negative isolates were further screened for their low susceptibility to β-lactamic agents, and found to belong to Pseudomonaceae family, with predominance of Pseudomonas putida (92 %). Minimal Inhibitory Concentration (MIC) was determined for five β-lactams and the distributive analysis of cefotaxime MIC performed, allowing the first report of Epidemiological Cut-OFF values for P. putida regarding such antibiotic. Hence, 46 % of the isolates from farms presented acquired resistance to cefotaxime, with fluvial strains presenting an acquisition of AMR in 22 % of the isolates. The response to β-lactams impact in P. putida is different from Pseudomonas aeruginosa’s, the family type strain, showing that data determined for a species should only be extended to other bacteria with caution, even closely related. It becomes crucial to broaden present research, mainly focused on few pathogenic bacteria, to other microorganisms carrying relevant resistance tools or capable of genetic transfer to more virulent strains. Most available data on AMR so far has been obtained from studies performed in restricted clinical or veterinary context, showing the result of a strong selective pressure related to therapy but often disregarding the origin of the AMR mechanisms encountered. The strong impact that environmental microorganisms have (and probably already had in the past) on the evolution and spreading of AMR, is just beginning to be unveiled.     

A phytogeographic analysis of cloud forests and other forest subtypes amidst the Atlantic forests in south and southeast Brazil

In a previous study near the summit of Mt. Cuscuzeiro (Ubatuba, SP) (820–1270 m), on the SE Brazilian coast, we found two floristically different forests, one above 1120 m, that appears to have a number of features typical of cloud forests, and another on the lower altitude slopes below. Taking these two forests as reference points, we addressed two questions: (1) What are their floristic relationships with other Atlantic forest subtypes in S-SE Brazil?; (2) Do the cloud forests in this region constitute a particular floristic-phytogeographic formation or are they a subset of their surrounding community? Species from 109 surveys (including Mount Cuscuzeiro) of 83 locations in S-SE Brazil were compiled into a binary (presence-absence) floristic matrix. Analyses of similarity among these samples using clustering (UPGMA, TWINSPAN) and ordination (DCA, PCO and CA) methods were performed. The surveys were divided into six main groups: (1) Cloud Forests; (2) “Salesópolis” group (3) Coastal Forests, subdivided between (a) Slope Forests and (b) Coastal Plain (“Restinga”) Forests and Mountaintop Forests (not included in the Cloud Forests group); (4) Araucaria Forests; (5) Inland Seasonal Forests (from below ca. 700 m); and (6) Inland Montane Forests (from above ca. 700 m). The preferential and indicator species of the Cloud Forest group produced by TWINSPAN are presented. The Mount Cuscuzeiro forests from above and from below 1120 m were clustered with the Cloud Forests and the coastal Slope Forests groups, respectively. We concluded that Cloud forests comprise a distinct phytogeographic formation in Brazilian S-SE region.     

Preparative chromatography for obtaining pure samples of rosaniline,magenta II,and new fuchsine

A simple low pressure liquid chromatographic method is reported that can separate the basic fuchsine homologues, rosaniline, magenta II and new fuchsine from an impure commercial dye. The chromatographic purity of the separated dyes is > 90%. All homologues were obtained in multi-milligram amounts per chromatographic run; precise yields depend on the composition of the starting material and potentially may be greater. This is a useful preparative procedure for generating chromatographically pure samples of basic fuchsine homologues, especially those that cannot be obtained in pure form by direct synthesis.     

ProDy: protein dynamics inferred from theory and experiments

SUMMARY: We developed a Python package, ProDy, for structure-based analysis of protein dynamics. ProDy allows for quantitative characterization of structural variations in heterogeneous datasets of structures experimentally resolved for a given biomolecular system, and for comparison of these variations with the theoretically predicted equilibrium dynamics. Datasets include structural ensembles for a given family or subfamily of proteins, their mutants and sequence homologues, in the presence/absence of their substrates, ligands or inhibitors. Numerous helper functions enable comparative analysis of experimental and theoretical data, and visualization of the principal changes in conformations that are accessible in different functional states. ProDy application programming interface (API) has been designed so that users can easily extend the software and implement new methods. AVAILABILITY: ProDy is open source and freely available under GNU General Public License from http://www.csb.pitt.edu/ProDy/.     

Kebab: kinetochore and EB1 associated basic protein that dynamically changes its localisation during Drosophila mitosis

Microtubule plus ends are dynamic ends that interact with other cellular structures. Microtubule plus end tracking proteins are considered to play important roles in the regulation of microtubule plus ends. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Here we report the identification of a novel Drosophila protein, which we call Kebab (kinetochore and EB1 associated basic protein), through in vitro expression screening for EB1-interacting proteins. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. In telophase, it associates with microtubules in central-spindle and centrosomal regions. The localisation to kinetochores depends on microtubules. The protein has a domain most similar to the atypical CH domain of Ndc80, and a coiled-coil domain. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localisation. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility.