Chemical Synthesis of a Full-Length G-Protein-Coupled Receptor ß2-Adrenergic Receptor with Defined Modification Patterns at the C-Terminus.

The ß2-adrenergic receptor (ß2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. ß2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length ß2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length ß2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length ß2ARs with distinct modification patterns, including a full-length ß2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length ß2AR receptors, we found that different phosphorylation patterns mediate different interactions with ß-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between ß2AR and ß-arrestin1. Access to full-length ß2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of ß2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

Prevalence and characterization of oxazolidinone and phenicol cross-resistance gene optrA in enterococci obtained from anaerobic digestion systems treating swine manure.

The use of the phenicol antibiotic florfenicol in livestock can select for the optrA gene, which also confers resistance to the critically important oxazolidinone antibiotic linezolid. However, the occurrence and dissemination of florfenicol and linezolid cross-resistance genes in anaerobic treatment systems for livestock waste are unknown. Herein, the phenotypes and genotypes (optrA, fexA, fexB, and cfr) of florfenicol and linezolid cross-resistance were investigated in 339 enterococci strains isolated from lab- and full-scale mesophilic anaerobic digestion systems treating swine waste. It was found that optrA, fexA, and fexB were frequently detected in isolated enterococci in both systems by PCR screening, whereas cfr was not detected. The most abundant gene was optrA, which was detected in 73.5% (n = 50) and 38.9% (n = 23) of enterococci isolates in the full-scale influent and effluent, respectively. Most strains carried more than two resistance genes, and the average percentage of co-occurrence of optrA/fexA was 16.6%. Based on minimum inhibitory concentrations of the enterococci strain phenotypes, 85.7%, 77.5%, and 77.5% of strains in influent were resistant to chloramphenicol, florfenicol, and linezolid, respectively, while 56.3%, 65.2%, and 13% in the effluent isolates were found, respectively, which was consistent with the genotype results. The phenotypes and genotypes of florfenicol and linezolid resistance were relative stable in the enterococci isolated from the influent and effluent in lab-scale anaerobic digestion system. The findings signify the enterococci isolates harboring the optrA gene remained in effluents of both full- and lab-scale swine waste anaerobic digestion system; hence, effective management strategies should be implemented to prevent the discharge of antibiotic resistance from the livestock waste treatment systems.

Influence of different substrates on the formation and characteristics of aerobic granules in sequencing batch reactors.

The effects of different substrates on the aerobic granulation process were studied using laboratory-scale sequencing batch reactors (SBRs). Four parallel granules sequencing batch reactors (GSBR): R1, R2, R3, and R4 were fed with acetate, glucose, peptone and fecula, respectively. Stable aerobic granules were successfully cultivated in R1, R2, R4, and smaller granules less than 500 microm were formed in R3. Morphology and the physic-chemical characteristics of aerobic granules fed with different carbon substrates were investigated by the four reactors operated under the same pressure. The aerobic granules in the four reactors were observed and found that peptone was the most stable one due to its good settleability even after a sludge age as short as 10 d. A strong correlation was testified between the characteristics of aerobic granules and the properties of carbon substrates. The stability of aerobic granules was affected by extracellular polymer substances (EPS) derived from microorganism growth during feast time fed with different carbon substrates, and the influence of the property of storage substance was greater than that of its quantity. Optimal carbon substrates, which are helpful in the cultivation and retention of well-settling granules and in the enhancement of the overall ability of the aerobic granules reactors, were found.

[Aerobic nitrifying granulation fed with source-separated urine in sequencing batch reactor].

Morphological characteristics of aerobic nitrifying granules that were utilized to treat the diluted source-separated urine were investigated in two lab-scale sequencing batch reactors. In the first sequencing batch reactor, which was inoculated with nitrifying bacteria, the COD of effluent was markedly decreased while the nitrification rate was very low. Aerobic nitrifying granules were not appeared in the first bioreactor. In the other SBR system that was inoculated with aerobic granules cultivated in the laboratory, the COD and ammonia in source-separated urine were effectively removed and the removal rate was more than 90%. Under operational condition that influent ammonia volume rate was 0.5 kg/(m3 x d) for 70 days, the aerobic nitrifying granules were stable in the reactor. Aerobic nitrifying granules have well settleability and metabolize activity, the surface of granules was occupied by nitrifying bacillus and cocci bacteria. Contrast to the inoculating aerobic granules, the diameter of aerobic nitrifying granules dramatically decreased to more than 2 mm and the settle velocity were greater than that of aerobic granules which have the same diameter.

[Characterization of bubble size distribution in ES-DAF unit].

ES-DAF unit was introduced and studied in this paper. Without a costly air saturator, ES-DAF consists of an ejector and a static mixer between the pressure side and suction side of the recycle rotary pump. The bubble size distribution in this novel unit was studied by using a CCD imagination through a microscope. The bubble size decreased with the increase of cycle ratio or the decrease of superficial air-water ratio. These results suggest that smaller bubbles would be formed when the initial number of nucleation sites increase by enhancing the turbulence intensity in the saturation system. The bubble size distribution became lower and wider with the increase of saturation pressure because of more frequent bubble collision and coalescence.