Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.     

Evidence of amelogenesis imperfecta in an early African Homo erectus

The teeth of the Homo erectus child (Garba IV) recovered from Melka Kunture Ethiopia and dated to 1.5 Ma are characterized by generalized enamel dysplasia, reduced enamel radio-opacity, and severe attrition. This combination of features is found in a large group of hereditary, generalized enamel dysplasias known as amelogenesis imperfecta (AI). SEM studies carried out on epoxy replicas of teeth from the Garba IV child, confirmed that the defects noted were developmental and not due to diagenesis. The enamel prism arrangement is abnormal and there are deep vertical furrows lacking enamel on both buccal and lingual surfaces of all molars. The lesions differ from those characteristic of linear enamel hypoplasia that form discrete horizontal lesions or pits within otherwise normal enamel. We propose that the Garba IV child is the earliest example of AI and provides a link between palaeoanthropology and molecular biology in investigations of the evolutionary history of genetic disorders.     

Genome-wide miRNA signatures of human longevity

Little is known about the functions of miRNAs in human longevity. Here, we present the first genome-wide miRNA study in long-lived individuals (LLI) who are considered a model for healthy aging. Using a microarray with 863 miRNAs, we compared the expression profiles obtained from blood samples of 15 centenarians and nonagenarians (mean age 96.4 years) with those of 55 younger individuals (mean age 45.9 years). Eighty miRNAs showed aging-associated expression changes, with 16 miRNAs being up-regulated and 64 down-regulated in the LLI relative to the younger probands. Seven of the eight selected aging-related biomarkers were technically validated using quantitative RT-PCR, confirming the microarray data. Three of the eight miRNAs were further investigated in independent samples of 15 LLI and 17 younger participants (mean age 101.5 and 36.9 years, respectively). Our screening confirmed previously published miRNAs of human aging, thus reflecting the utility of the applied approach. The hierarchical clustering analysis of the miRNA microarray expression data revealed a distinct separation between the LLI and the younger controls (P-value < 10(-5) ). The down-regulated miRNAs appeared as a cluster and were more often reported in the context of diseases than the up-regulated miRNAs. Moreover, many of the differentially regulated miRNAs are known to exhibit contrasting expression patterns in major age-related diseases. Further in silico analyses showed enrichment of potential targets of the down-regulated miRNAs in p53 and other cancer pathways. Altogether, synchronized miRNA-p53 activities could be involved in the prevention of tumorigenesis and the maintenance of genomic integrity during aging.     

Role of miRNA and miRNA processing factors in development and disease

Mature microRNAs (miRNAs) are single-stranded RNA molecules of 17-24 nucleotides (nt) in length that are encoded in the genomes of plants and animals. The seminal discoveries of miRNA made in C. elegans have led the way to the rampant discoveries being made today in this field. Since each miRNA is predicted and in some cases confirmed to regulate multiple genes, the potential regulatory circuitry afforded by miRNAs is thought to be enormous and could amount to regulation of >30% of all human genes. Due to the sequences of many of the miRNAs being highly homologous among organisms, the huge potential of miRNAs to regulate gene expression, and the hints of miRNAs being useful in both diagnostics and therapeutics, it is no wonder these small RNAs are gaining such popularity in both the academic and industrial settings. It is now becoming clear that the miRNA gene class represents a very important gene regulatory network. This article reviews the initial discoveries of miRNA that began in the nematode C. elegans, and extends into what is known about miRNAs and miRNA processing factors in mouse development and human disease.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

The use of miRNA microarrays for the analysis of cancer samples with global miRNA decrease

Recent studies have established that mutations or deletions in microRNA (miRNA) processing enzymes resulting in a global decrease of miRNA expression are frequent across cancers and can be associated with a poorer prognosis. While very popular in miRNA profiling studies, it remains unclear whether miRNA microarrays are suited or not to accurately detecting global miRNA decreases seen in cancers. In this work, we analyzed the miRNA profiles of samples with global miRNA decreases using Affymetrix miRNA microarrays following the inducible genetic deletion of Dicer1. Surprisingly, up to a third of deregulated miRNAs identified upon Dicer1 depletion were found to be up-regulated following standard robust multichip average (RMA) background correction and quantile normalization, indicative of normalization bias. Our comparisons of five preprocess steps performed at the probe level demonstrated that the use of cyclic loess relying on non-miRNA small RNAs present on the Affymetrix platform significantly improved specificity and sensitivity of detection of decreased miRNAs. These findings were validated in samples from patients with prostate cancer, where conjugation of robust normal-exponential background correction with cyclic loess normalization and array weights correctly identified the greatest number of decreased miRNAs, and the lowest amount of false-positive up-regulated miRNAs. These findings highlight the importance of miRNA microarray normalization for the detection of miRNAs that are truly differentially expressed and suggest that the use of cyclic loess based on non-miRNA small RNAs can help to improve the sensitivity and specificity of miRNA profiling in cancer samples with global miRNA decrease.     

Subtyping glioblastoma by combining miRNA and mRNA expression data using compressed sensing-based approach

In the clinical practice, many diseases such as glioblastoma, leukemia, diabetes, and prostates have multiple subtypes. Classifying subtypes accurately using genomic data will provide individualized treatments to target-specific disease subtypes. However, it is often difficult to obtain satisfactory classification accuracy using only one type of data, because the subtypes of a disease can exhibit similar patterns in one data type. Fortunately, multiple types of genomic data are often available due to the rapid development of genomic techniques. This raises the question on whether the classification performance can significantly be improved by combining multiple types of genomic data. In this article, we classified four subtypes of glioblastoma multiforme (GBM) with multiple types of genome-wide data (e.g., mRNA and miRNA expression) from The Cancer Genome Atlas (TCGA) project. We proposed a multi-class compressed sensing-based detector (MCSD) for this study. The MCSD was trained with data from TCGA and then applied to subtype GBM patients using an independent testing data. We performed the classification on the same patient subjects with three data types, i.e., miRNA expression data, mRNA (or gene expression) data, and their combinations. The classification accuracy is 69.1% with the miRNA expression data, 52.7% with mRNA expression data, and 90.9% with the combination of both mRNA and miRNA expression data. In addition, some biomarkers identified by the integrated approaches have been confirmed with results from the published literatures. These results indicate that the combined analysis can significantly improve the accuracy of classifying GBM subtypes and identify potential biomarkers for disease diagnosis.     

玉米Dof转录因子家族基因的全基因组分析

Dof转录因子家族在植物生长发育和基因表达调控过程中具有重要的作用,本文利用公布的玉米基因组草图数据,利用生物信息学方法对玉米全基因组Dof基因的结构、系统进化关系和保守motif进行了分析。结果表明:玉米中共有18个Dof类型基因,命名为ZmDof1-ZmDof18,其蛋白质长度在211aa至618aa之间,通过系统进化树分析后,18个Dof基因可以明显的分为三类,此外玉米Dof基因的数目远远小于水稻和拟南芥,基因复制现象较少是玉米Dof基因数量较少的原因之一,MEME分析证实了Dof基因含有三个保守的motif。对玉米Dof类型基因的系统分析,将有助于玉米Dof类型基因的克隆和功能的进一步研究。     

牦牛NLRP基因家族全基因组鉴定及转录组分析

NLRP基因家族在哺乳动物基因组中分布广泛,在免疫应答和生殖相关过程中发挥重要作用。本研究根据已报道的人类NLRP基因,利用PFAM、CDD、UniProt等数据库,采用ClustalW、MEGA X、ExPASy、MEME、GSDS和STRING等软件对牦牛NLRP家族成员进行全基因组鉴定和分析,并且基于高通量测序数据分析NLRP基因在牦牛不同组织中的表达情况。研究共鉴定出8个不同的NLRP基因家族成员,包括NLRP2、NLRP3、NLRP5、NLRP6、NLRP8、NLRP9、NLRP12、NLRP14,其中NLRP9有2个可变剪切。牦牛NLRP蛋白均有多个磷酸化位点,GRAVY值均小于0,属于亲水性蛋白;不稳定系数均大于40,均为不稳定蛋白,无跨膜结构。系统进化树显示同一家族成员氨基酸序列相似性高,在 6个物种中都聚在一起,免疫相关基因与生殖发育相关基因在进化中已经分开。蛋白互作网络显示牦牛NLRP蛋白与其他物种已经研究的功能相似,不仅与参与免疫相关的蛋白互作,也与生殖标记蛋白互作。如NLRP3与多种参与炎症反应的蛋白PYCARD、MLKL、GSDMD互作,NLRP5也与生殖细胞标记蛋白GDF9、BMP15、ZAR1等互作。转录组分析结果显示,牦牛NLRP基因在6种组织中的表达较为微弱或者不表达,具有组织特异性。牦牛NLRP家族在结构及理化性质上与其他物种同源基因具有相同的特点,部分试验和预测证实其同样参与了免疫和生殖相关过程。本研究结论不仅对牦牛NLRP基因家族进行了系统分析,同时为进一步研究牦牛先天免疫和生殖相关过程提供理论依据。     

Computational analysis of drought stress-associated miRNAs and miRNA co-regulation network in Physcomitrella patens

miRNAs are non-coding small RNAs that involve diverse biological processes. Until now, little is known about their roles in plant drought resistance. Physcomitrella patens is highly tolerant to drought; however, it is not clear about the basic biology of the traits that contribute P. patens this important character. In this work, we discovered 16 drought stress-associated miRNA (DsAmR) families in P. patens through computational analysis. Due to the possible discrepancy of expression periods and tissue distributions between potential DsAmRs and their targeting genes, and the existence of false positive results in computational identification, the prediction results should be examined with further experimental validation. We also constructed an miRNA co-regulation network, and identified two network hubs, miR902a-5p and miR414, which may play important roles in regulating drought-resistance traits. We distributed our results through an online database named ppt-miRBase, which can be accessed at http://bioinfor.cnu.edu.cn/ppt_miRBase/index.php. Our methods in finding DsAmR and miRNA co-regulation network showed a new direction for identifying miRNA functions.     

miRNA*生物合成及其功能研究的新发现

miRNA*是在miRNA加工成熟过程中与其互补的大约22个核苷酸的RNA序列。传统观点认为miRNA*是miRNA生物合成中形成的没有功能的副产品。然而最近的研究发现miRNA*s与miRNA一样, 主要介导转录后的基因调控网络; 但不同的是, miRNA与Argonaute1蛋白(AGO1)结合形成RNA诱导的沉默复合体(RISC), 而miRNA*却在AGO2的帮助下形成RISC复合体进行RNA干涉, 这点与siRNA的作用方式类似。文章从miRNA*的生物合成、生物学特性和功能等方面综述了miRNA*最新研究进展。