Complexity and Specificity of Precursor microRNAs Driven by Transposable Elements in Rice

In eukaryotes, small noncoding RNA molecules of 16–29 nucleotides in length play crucial roles in the regulation of gene expression. Some 377 sequences representing rice pseudo-microRNAs (miRNAs) are available in release 13.0 of the miRBase sequence database () and are grouped into 143 families. Most newly deposited miRNA sequences are likely to be species-specific. To understand the relationship between miRNAs and transposable elements (TEs) in rice, the RepeatMasker application () was used to screen single-stranded precursor miRNA (pre-miRNA) sequences. This analysis revealed that 33.1% of miRNAs and 36.4% of miRNA families are associated with interspersed repeats, and most of them are species-specific. Furthermore, multiple miRNA families can be encoded by the same TE class. Alignment analysis revealed that miR439 originated from an MuDR4-OS TE, which amplified and diversified in the genome as an inverted repeat of the core sequence followed by multiple repeats. Multiple copies of miR445 and its complexity originate from and are driven by the DNA/Tourist TE class. These results provide an important contribution to the elucidation of TE-driven mechanisms that regulate the species specificity and complexity of rice miRNAs.     

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.     

Antioxidant and antiproliferative activities of hydroxyl-substituted Schiff bases

Eight hydroxyl-substituted Schiff bases with the different number and position of hydroxyl group on the two asymmetric aromatic rings (A and B rings) were prepared by the reaction between the corresponding aromatic aldehyde and aniline. Their antioxidant effects against the stable galvinoxyl radical (GO) in ethyl acetate and methanol, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced DNA strand breakage, and their antiproliferative effects on human hepatoma HepG2 cells, were investigated. Structure–activity relationship analysis demonstrates that o-dihydroxyl groups on the aromatic A ring and 4-hydroxyl group attached to the aromatic B ring contribute critically to the antioxidant and antiproliferative activities.     

Stereochemistry–activity relationship of orally active tetralin S1P agonist prodrugs

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (?)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.     

Isoform-selective inhibition of chrysin towards human cytochrome P450 1A2. Kinetics analysis,molecular docking,and molecular dynamics simulations

Our kinetics studies demonstrated that the nature product chrysin exhibited a high inhibitory affinity of 54 nM towards human cytochrome P450 1A2 and was comparable to α-naphthoflavone (49 nM), whereas it represented a moderate affinity of 5225 nM against human cytochrome P450 2C9. However, it remains unclear how this inhibitor selectively binds 1A2. To better understand the isoform selectivity of chrysin, molecular docking and molecular dynamics simulations were performed. Chrysin formed a strong H-bond with Asp313 of 1A2. The stacking interactions with Phe226 also contributed to its tight binding to 1A2. The larger and much more open active site architectures of 2C9 may explain the weaker inhibitory affinity of chrysin towards 2C9. The predicted binding free energies suggest that chrysin preferred 1A2 (ΔG_{bind, pred} = ?23.11 kcal/mol) to 2C9 (?20.41 kcal/mol). Additionally, the present work revealed that 7-hydroxy-flavone bound to 1A2 in a similar pattern as chrysin and represented a slightly less negative predicted binding free energy, which was further validated by our kinetics analysis (IC₅₀ = 240 nM). Results of the study can provide insight for designing novel isoform-selective 1A2 inhibitors.     

A Novel Statistic for Genome-Wide Interaction Analysis

Although great progress in genome-wide association studies (GWAS) has been made, the significant SNP associations identified by GWAS account for only a few percent of the genetic variance, leading many to question where and how we can find the missing heritability. There is increasing interest in genome-wide interaction analysis as a possible source of finding heritability unexplained by current GWAS. However, the existing statistics for testing interaction have low power for genome-wide interaction analysis. To meet challenges raised by genome-wide interactional analysis, we have developed a novel statistic for testing interaction between two loci (either linked or unlinked). The null distribution and the type I error rates of the new statistic for testing interaction are validated using simulations. Extensive power studies show that the developed statistic has much higher power to detect interaction than classical logistic regression. The results identified 44 and 211 pairs of SNPs showing significant evidence of interactions with FDR<0.001 and 0.001<FDR<0.003, respectively, which were seen in two independent studies of psoriasis. These included five interacting pairs of SNPs in genes LST1/NCR3, CXCR5/BCL9L, and GLS2, some of which were located in the target sites of miR-324-3p, miR-433, and miR-382, as well as 15 pairs of interacting SNPs that had nonsynonymous substitutions. Our results demonstrated that genome-wide interaction analysis is a valuable tool for finding remaining missing heritability unexplained by the current GWAS, and the developed novel statistic is able to search significant interaction between SNPs across the genome. Real data analysis showed that the results of genome-wide interaction analysis can be replicated in two independent studies.     

Polymorphisms of the DNA repair gene MGMT and risk and progression of head and neck cancer

Methylating agents are involved in carcinogenesis, and the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) removes methyl group from O⁶-methylguanine. Genetic variation in DNA repair genes has been shown to contribute to susceptibility to squamous cell carcinoma of the head and neck (SCCHN). We hypothesize that MGMT polymorphisms are associated with risk of SCCHN. In a hospital-based case–control study of 721 patients with SCCHN and 1234 cancer-free controls frequency-matched by age, sex and ethnicity, we genotyped four MGMT polymorphisms, two in exon 3, 16195C > T and 16286C > T and two in the promoter region, 45996G > T and 46346C > A. We found that none of these polymorphisms alone had a significant effect on risk of SCCHN. However, when these four polymorphisms were evaluated together by the number of putative risk genotypes (i.e. 16195CC, 16286CC, 45996GT + TT, and 46346CA + AA), a statistically significantly increased risk of SCCHN was associated with the combined genotypes with three to four risk genotypes, compared with those with zero to two risk genotypes (adjusted odds ratio (OR) = 1.27; 95% confidence interval (CI) = 1.05–1.53). This increased risk was also more pronounced among young subjects (OR = 1.81; 95% CI = 1.11–2.96), men (OR = 1.24; 95% CI = 1.00–1.55), ever smokers (OR = 1.25; 95% = 1.01–1.56), ever drinkers (OR = 1.29; 95% CI = 1.04–1.60), patients with oropharyngeal cancer (OR = 1.45; 95% CI = 1.12–1.87), and oropharyngeal cancer with regional lymph node metastasis (OR = 1.52; 95% CI = 1.16–1.89). In conclusion, our results suggest that any one of MGMT variants may not have a substantial effect on SCCHN risk, but a joint effect of several MGMT variants may contribute to risk and progression of SCCHN, particularly for oropharyngeal cancer, in non-Hispanic whites.     

In Vitro Degradation of Insulin-like Peptide 3 by Insulin-degrading Enzyme

Insulin-like peptide 3 (INSL3) is an insulin superfamily peptide hormone, primarily expressed in the testes and playing a key role in the fetus testes descent and suppression of male germ cell apoptosis. Insulin-degrading enzyme (IDE) is a zinc-metalloprotease, responsible for in vivo degradation of insulin, Aβ, and other peptide hormones. IDE has high expression level in the testes, implying it might be involved in INSL3 turnover in vivo. In present work, we studied in vitro degradation of INSL3 by IDE. Recombinant human IDE degraded human INSL3, but its degradation rate for INSL3 is more than a magnitude lower than that for insulin. However, IDE bound INSL3 and insulin with almost same affinity. IDE cleaved the peptide bond between B26R and B27W of INSL3, and released a pentapeptide, WSTEA, from the C-terminal of B-chain. Our present work suggested that IDE might play a role in INSL3 degradation in vivo.