A Set of Plasmid-Based Modules for Easy Switching of C-Terminal Epitope Tags in Saccharomyces cerevisiae

Epitope tagging is a powerful strategy for analyzing the functions of targeted proteins. The use of this strategy has become more convenient with the development of the epitope switch, which is another type of epitope tagging designed to convert the previously tagged epitopes on the chromosome to other epitopes of interest. Various modules for C-terminal epitope switching have been developed and amplified using the one-step polymerase chain reaction (PCR) method before transformation. However, PCR amplification occasionally generates mutations that affect the fidelity of epitope switching. Here, we constructed several plasmids to isolate modules for epitope switching through digestion by restriction enzymes. The isolated modules contained DNA sequences for homologous recombination, various epitopes (13×Myc, 6×HA, GFP, Venus, YFP, mCherry, and CFP), and a transformation marker (Candida glabrata LEU2). The restriction enzyme-digested plasmids were used to directly transform the cells for epitope switching. We demonstrate the efficient and accurate switching of the MX6 module-based C-terminal tandem affinity purification tags to each aforementioned epitope. We believe that our plasmids can serve as powerful tools for the functional analysis of yeast proteins.

Epidemiologic Features and Influencing Factors of Norovirus Outbreaks in the City of Wuxi, China from 2014 to 2018

The study investigated the genotypic changes and epidemiologic features of norovirus outbreaks and factors influencing the attack rate and outbreak duration in Wuxi from 2014 to 2018. Norovirus outbreaks, monitored through surveillance system, were investigated. The norovirus-positive specimens from outbreaks were collected and genotyped using a dual polymerase-capsid genotyping protocol based on a one-step polymerase chain reaction (PCR) amplicon. The genotypes were analyzed by Norovirus Typing Tool Version 2.0. A total of 74 norovirus outbreaks were reported in Wuxi from 2014 to 2018. Most (93.2%) norovirus outbreaks were caused by GII genotypes. The predominant norovirus genotypes in outbreaks have changed from GII.17 (20.3%) in 2014–2015 to GII.P16/GII.2 (40.5%) in 2017–2018. GII.P16/GII.2 in 2017–2018 season were more prevalent than GII.17 in 2014–2015 season (χ2 = 4.741, P = 0.029). 56.7% of the outbreaks occurred in primary schools. The re-outbreak rate was 16.2%. 66.7% of re-outbreaks were caused by norovirus variants different from previous genotypes. Outbreaks in nonprimary school settings (odds ratio [OR]: 4.007; 95% CI: 1.247–12.876) and those leading to temporary school or institution closure (OR: 20.510; 95% CI: 1.806–232.937) were reported with a higher attack rate. The outbreaks in primary schools (OR: 4.248; 95% CI: 1.211–14.903), re-outbreaks (OR: 6.433; 95% CI: 1.103–37.534) and longer report timing (OR: 8.380; 95% CI: 2.259–31.089) declared a significantly longer duration. It is of great importance that the monitoring of norovirus outbreaks for the emergence of novel strains, along with responsive prevention and control intervention should be strengthened in adults and school-age population, especially in primary students and preschool children.

Biochip-based approach for comprehensive pharmacogenetic testing

ObjectivesIndividual sensitivity to many widely used drugs is significantly associated with genetic factors. The purpose of our work was to develop an instrument for simultaneous determination of the most clinically relevant pharmacogenetic markers to allow personalized treatment, mainly in patients with cardiovascular diseases.MethodsMultiplex one-step polymerase chain reaction (PCR) followed by hybridization on a low-density biochip was applied to interrogate 15 polymorphisms in the following eight genes: VKORC1 –1639 G>A, CYP4F2 1297 G>A, GGCX 2374 C>G, CYP2C9 *2,*3 (430 C>T, 1075 A>C), CYP2D6 *3,*4, *6, *9, *41 (2549delA, 1846 G>A, 1707delT, 2615_2617delAAG, 2988 G>A), CYP2C19 *2,*3,*17 (681 G>A, 636 G>A, −806 C>T), ABCB1 (3435 C>T), SLCO1B1 *5.ResultsTwo hundred nineteen patients with cardiovascular diseases (CVD) and 48 female patients with estrogen receptor (ER)-positive breast cancer (BC) were genotyped. Of the 219 CVD patients, 203 (92.7%) carried one or more actionable at-risk genotypes based on VKORC1/CYP2C9, CYP2C9, CYP2C19, SLCO1B1, and CYP2D6 genotypes. Among them, 67 patients (30.6%) carried one, 58 patients (26.5%) carried two, 51 patients (23.3%) carried three, 26 patients (11.9%) carried four, and one patient (0.4%) carried five risk actionable genotypes. In the ER-positive BC group 12 patients (25%) were CYP2D6 intermediate or poor metabolizers.ConclusionsThe developed biochip is applicable for rapid and robust genotyping of patients who were taking a wide spectrum of medications to optimize drugs and dosage and avoid adverse drug reactions in cardiology, oncology, psychiatry, rheumatology and gastroenterology.