Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.     

Association between Tumor Necrosis Factor-α 308G/A Gene Polymorphism and Silicosis Susceptibility: A Meta-Analysis

Background

Tumor necrosis factor-α (TNF-α) 308 G/A gene polymorphism has been reported to be associated with susceptibility to silicosis. However, the relevant study results are still inconsistent.

Objective and Methods

A meta-analysis was performed in order to drive a more precise estimation of the relationship between TNF-α-308 G/A gene polymorphism and susceptibility to silicosis. Electronic databases were searched and nine separate studies were included. The pooled odds ratios (ORs) and the corresponding 95% confidence internal (CI) were calculated by a fixed effect model.

Results

A total of 1267 cases and 1214 controls were included. In the overall analysis, significantly increased silicosis risk was found (for GA+AA vs. GG OR=1.45, 95%CI: 1.20-1.760, P=1.58E4; for GA vs. GG: OR=1.53, 95%CI=1.25-1.86, P=3.11E5; for A allele vs. G allele: OR=1.27, 95%CI=1.08-1.50, P= 0.004). In the subgroup analysis, significantly increased silicosis risk was also found among Asians (for GA+AA vs. GG: OR=1.63, 95%CI=1.27-2.08, P=1.01E4), for GA vs. GG: OR=1.71, 95%CI=1.33-2.20, P=3.44E5), for A allele vs. G allele: OR=1.45, 95%CI=1.17-1.80, P=0.001). However, no significantly increased risk was found among non-Asians for all genetic models.

Conclusions

TNF-α-308 G/A polymorphism might lead to an increased risk of silicosis susceptibility, especially for Asians. However, further studies with large sample sizes should be conducted to confirm the association.     

Enhanced aerobic granulation and nitrogen removal by the addition of zeolite powder in a sequencing batch reactor

The aim of this study was to evaluate the impact of zeolite powders on feasibility of rapid aerobic granulation in the column-type sequencing batch reactors. After 90 days' operation, aerobic granular sludge was formed in both reactors by altering influent chemical oxygen demand/nitrogen (COD/N) ratios. R1 with zeolite powders had better removal capabilities of COD and total nitrogen than R2, which was without zeolite powders. Mixed liquor volatile suspended solid concentrations of the two reactors were 7.36 and 5.45 g/L, while sludge volume index (SVI₃₀) values were 34.9 and 47.9 mg/L, respectively. The mean diameters of aerobic granular sludge in the above two reactors were 2.5 and 1.5 mm, respectively. Both reactors achieved the largest simultaneous nitrification and denitrification (SND) efficiency at an influent COD/N ratio of 8; however, R1 exhibited more excellent SND efficiency than R2. The obtained results could provide a novel technique for rapid aerobic granulation and N removal simultaneously, especially when treating nitrogen-rich industrial wastewater.     

Oxygen isotope temperature calibrations for modern Tridacna shells in western Pacific

Coral Reefs - The oxygen isotope ratio of carbonate in Tridacna shell (δ18Oshell) is assumed to be precipitated in isotopic equilibrium with surrounding seawater and thus reflects a...     

Protocadherin 2C: a new member of the protocadherin 2 subfamily expressed in a redundant manner with OL-protocadherin in the developing brain.

Using cDNA of human protocadherin 2A (pc2A; originally known as protocadherin 2) as a probe, we cloned a new member of the protocadherin 2 subfamily from mouse brain cDNA libraries and named it protocadherin 2C (pc2C). It was similar to pc2A throughout its entire coding region, and its C-terminal region was highly conserved. The locus of the pc2C gene was on the mouse chromosome 18C where the pc2A gene is located, suggesting that genes of the pc2 subfamily form a gene cluster. The expression of pc2C was restricted to the nervous system, and the expression started in the embryonic stage and increased up to the adult stage. The expression pattern was quite similar to that of OL-protocadherin, a distinct class of protocadherin, although the timing and relative strength of expression were different. These results suggest that pc2C may be involved in neural development along with other classes of protocadherins.     

Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice

The mechanism(s) by which vitamin D(3) regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D(3) deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D(3)) null mice to study the effect of vitamin D(3) deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D(3)-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D(3) deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D(3)-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D(3)-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D(3)-replete diet. Estrous cycles were restored when vitamin D(3)-deficient Cyp27b1 null young adult females were transferred to a vitamin D(3)-replete diet. This study is the first to demonstrate that peripubertal vitamin D(3) sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D(3) is a key regulator of neuroendocrine and ovarian physiology.     

Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

Clear cell renal cell carcinoma (ccRCC) is a primary kidney cancer with high aggressive phenotype and extremely poor prognosis. Accumulating evidence suggests that circular RNAs (circRNAs) play pivotal roles in the occurrence and development of various human cancers. However, the expression, clinical significance and regulatory role of circRNAs in ccRCC remain largely unclear. Here we report that circDVL1 to be reduced in the serums and tissues from ccRCC patients, and to negatively correlate with ccRCC malignant features. Overexpression of circDVL1 inhibits proliferation, induces G1/S arrest, triggers apoptosis, and reduces migration and invasion in different ccRCC cells in vitro. Correspondingly, circDVL1 overexpression suppresses ccRCC tumorigenicity in a mouse xenograft model. Mechanistically, circDVL1 serves as a sponge for oncogenic miR-412-3p, thereby preventing miR-412-3p-mediated repression of its target protocadherin 7 (PCDH7) in ccRCC cells. Collectively, our results demonstrate that circDVL1 exerts tumor-suppressive function during ccRCC progression through circDVL1/miR-412-3p/PCDH7 axis, and suggest that circDVL1 could be a novel diagnostic and prognositc marker and therapeutic target for ccRCC.