Comparison of protein quantification and extraction methods suitable for E. coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations Sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and Sonication-BCA_P combinations.     

Chronic exposure to ozone induces cardiac antioxidant response and overexpression of either mitochondrial fision protein DRP1 and hipertrophyc-related proteins

Journal of Bioenergetics and Biomembranes - Pollution is considered a risk factor for cardiovascular disease; however, the mechanisms to explain this relationship are not well understood; ozone is...     

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ～10 nt, but it was required when flaps were ～500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ～20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ～20 nt and is completely required at ～50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ～20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.     

Inputs of seabird guano alter microbial growth,community composition and the phytoplankton–bacterial interactions in a coastal system

Seabird guano enters coastal waters providing bioavailable substrates for microbial plankton, but their role in marine ecosystem functioning remains poorly understood. Two concentrations of the water soluble fraction (WSF) of gull guano were added to different natural microbial communities collected in surface waters from the Ría de Vigo (NW Spain) in spring, summer, and winter. Samples were incubated with or without antibiotics (to block bacterial activity) to test whether gull guano stimulated phytoplankton and bacterial growth, caused changes in taxonomic composition, and altered phytoplankton–bacteria interactions. Alteromonadales, Sphingobacteriales, Verrucomicrobia and diatoms were generally stimulated by guano. Chlorophyll a (Chl a) concentration and bacterial abundance significantly increased after additions independently of the initial ambient nutrient concentrations. Our study demonstrates, for the first time, that the addition of guano altered the phytoplankton–bacteria interaction index from neutral (i.e. phytoplankton growth was not affected by bacterial activity) to positive (i.e. phytoplankton growth was stimulated by bacterial activity) in the low-nutrient environment occurring in spring. In contrast, when environmental nutrient concentrations were high, the interaction index changed from positive to neutral after guano additions, suggesting the presence of some secondary metabolite in the guano that is needed for phytoplankton growth, which would otherwise be supplied by bacteria.