Role of disulfide bridges in archaeal family-B DNA polymerases

The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.     

Treatment of estrogens and androgens in dairy wastewater by a constructed wetland system

Constructed wetland systems (CWS) have been used as a low cost bio-filtration system to treat farm wastewater. While studies have shown that CWS are efficient in removing organic compounds and pathogens, there is limited data on the presence of hormones in this type of treatment system.The objective of this study was to evaluate the ability of the CWS to reduce estrogenic and androgenic hormone concentration in dairy wastewater. This was achieved through a year long study on dairy wastewater samples obtained from a surface flow CWS. Analysis of hormonal levels was performed using a solid phase extraction (SPE) sample clean-up method, combined with reporter gene assays (RGAs) which incorporate relevant receptors capable of measuring total estrogenic or androgenic concentrations as low as 0.24 ng L⁻¹ and 6.9 ng L⁻¹ respectively. Monthly analysis showed a mean removal efficiency for estrogens of 95.2%, corresponding to an average residual concentration of 3.2 ng L⁻¹ 17β-estradiol equivalent (EEQ), below the proposed lowest observable effect concentration (LOEC) of 10 ng L⁻¹. However, for one month a peak EEQ concentration of 115 ng L⁻¹ was only reduced to 18.8 ng L⁻¹. The mean androgenic activity peaked at 360 ng L⁻¹ and a removal efficiency of 92.1% left an average residual concentration of 32.3 ng L⁻¹ testosterone equivalent (TEQ).The results obtained demonstrate that this type of CWS is an efficient system for the treatment of hormones in dairy wastewater. However, additional design improvements may be required to further enhance removal efficiency of peak hormone concentrations.     

First record of the non-indigenous parasitic copepod Neoergasilus japonicus (Harada, 1930) in the Lake Ontario Watershed: Oneida Lake,New York

Four specimens of the Asiatic parasitic copepod Neoergasilus japonicus (Harada, 1930) were collected from Oneida Lake, New York in September 2018; one specimen was from a white sucker Catostomus commersonii, another from a green sunfish Lepomis cyanellus, and two from a bluegill Lepomis macrochirus. The four adult female specimens were found attached to the base of the gills of their respective hosts along with other ergasilid species. The average total length of the adult female N. japonicus specimens we found was 0.609 mm. These detections represent the first known occurrence of this non-native species in the state of New York, extends the easternmost distribution of this parasite over 400 miles, and now includes the Lake Ontario watershed for the first time. It is commonly believed that the international aquaculture industry and aquarium fish trade are the most likely vectors of dispersal for N. japonicus. Monitoring the spread of non-indigenous aquatic species is an important step towards the development of management plans and mitigation efforts with regards to the anthropogenic causes of dispersal, and fish parasites are no exception.