Inhibition of the miR-17-92 Cluster Separates Stages of Palatogenesis.

The role that noncoding regions of the genome play in the etiology of cleft palate is not well studied. A novel method of microRNA (miR) inhibition that allows for specific miR knockdown in vivo has been developed by our laboratory. To further understand the role of miRs in palatogenesis, we used a new mouse model to inhibit specific miRs within the miR-17-92 cluster. Transgenic mice expressing inhibitory complexes for miR-17 and miR-18 manifested a clefting phenotype that was distinct from that observed in mice carrying inhibitory complexes for miR-17, miR-18, miR-19, and miR-92. An in silico candidate gene analysis and bioinformatics review led us to identify TGFBR2 as a likely target of miR-17 and miR-19 family members. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TGFBR1 and TGFBR2 expression levels were elevated in the palates of these miR transgenic embryos at embryonic day 15.5. RT-PCR data also showed that the expression of mature miRs from the miR-17-92 cluster was significantly decreased in the transgenic embryos. Decreased expression of TGFB pathway signaling ligands was also observed. Experiments in cells showed that inhibition of miR-17 and miR-18 was sufficient to induce increases in expression of TGFB receptors, while a concomitant decrease in TGFB signaling ligands was not observed. RT-PCR of mature miR-17-92 in cells demonstrated the selectivity and specificity of inhibitory complexes. While this study builds on previous studies that have implicated miR-17-92 in the regulation of important molecular components of the TGFB signaling pathway, it is likely that interactions remain to be elucidated between miR-17-92 and as-of-yet unidentified molecules important for the control of palatogenesis. The differential regulation of palatogenesis by members of the miR-17-92 cluster indicates that several gene combinations regulate palate elevation and extension during development.

Travel and Border Health Measures to Prevent the International Spread of Ebola.

During the 2014-2016 Ebola virus disease (Ebola) epidemic in West Africa, CDC implemented travel and border health measures to prevent international spread of the disease, educate and protect travelers and communities, and minimize disruption of international travel and trade. CDC staff provided in-country technical assistance for exit screening in countries in West Africa with Ebola outbreaks, implemented an enhanced entry risk assessment and management program for travelers at U.S. ports of entry, and disseminated information and guidance for specific groups of travelers and relevant organizations. New and existing partnerships were crucial to the success of this response, including partnerships with international organizations, such as the World Health Organization, the International Organization for Migration, and nongovernment organizations, as well as domestic partnerships with the U.S. Department of Homeland Security and state and local health departments. Although difficult to assess, travel and border health measures might have helped control the epidemic's spread in West Africa by deterring or preventing travel by symptomatic or exposed persons and by educating travelers about protecting themselves. Enhanced entry risk assessment at U.S. airports facilitated management of travelers after arrival, including the recommended active monitoring. These measures also reassured airlines, shipping companies, port partners, and travelers that travel was safe and might have helped maintain continued flow of passenger traffic and resources needed for the response to the affected region. Travel and border health measures implemented in the countries with Ebola outbreaks laid the foundation for future reconstruction efforts related to borders and travel, including development of regional surveillance systems, cross-border coordination, and implementation of core capacities at designated official points of entry in accordance with the International Health Regulations (2005). New mechanisms developed during this response to target risk assessment and management of travelers arriving in the United States may enhance future public health responses. The activities summarized in this report would not have been possible without collaboration with many U.S. and international partners (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/partners.html).

A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.

Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas-mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR family members because of duplications. Here, we report a novel plasmid-based miR inhibitor system (PMIS) that expresses a new RNA molecule, which inhibits miR family members in cells and mice. The PMIS engineered RNA optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. The PMIS shows no off-target effects or toxicity and is highly specific for miRs sharing identical seed sequences. Transgenic mice expressing both PMIS-miR-17-18 and PMIS-miR-19-92 show similar phenotypes of miR-17-92-knockout mice. Interestingly, mice only expressing PMIS-miR-17-18 have developmental defects distinct from mice only expressing PMIS-miR-19-92 demonstrating usefulness of the PMIS system to dissect different functions of miRs within clusters. Different PMIS miR inhibitors can be linked together to knock down multiple miRs expressed from different chromosomes. Inhibition of the miR-17-92, miR-106a-363 and miR-106b-25 clusters reveals new mechanisms and developmental defects for these miRs. We report a new tool to dissect the role of miRs in development without genome editing, inhibit miR function in cells and as a potential new therapeutic reagent.

The ontogeny of the chin: an analysis of allometric and biomechanical scaling.

The presence of a prominent chin in modern humans has been viewed by some researchers as an architectural adaptation to buttress the anterior corpus from bending stresses during mastication. In contrast, ontogenetic studies of mandibular symphyseal form suggest that a prominent chin results from the complex spatial interaction between the symphysis and surrounding soft tissue and skeletal anatomy during development. While variation in chin prominence is clearly influenced by differential growth and spatial constraints, it is unclear to what degree these developmental dynamics influence the mechanical properties of the symphysis. That is, do ontogenetic changes in symphyseal shape result in increased symphyseal bending resistance? We examined ontogenetic changes in the mechanical properties and shape of the symphysis using subjects from a longitudinal cephalometric growth study with ages ranging from 3 to 20+ years. We first examined whether ontogenetic changes in symphyseal shape were correlated with symphyseal vertical bending and wishboning resistance using multivariate regression. Secondly, we examined ontogenetic scaling of bending resistance relative to bending moment arm lengths. An ontogenetic increase in chin prominence was associated with decreased vertical bending resistance, while wishboning resistance was uncorrelated with ontogenetic development of the chin. Relative to bending moment arm lengths, vertical bending resistance scaled with significant negative allometry whereas wishboning resistance scaled isometrically. These results suggest a complex interaction between symphyseal ontogeny and bending resistance, and indicate that ontogenetic increases in chin projection do not provide greater bending resistance to the mandibular symphysis.

Candidate Gene Analyses of Skeletal Variation in Malocclusion.

This study evaluated associations between craniofacial candidate genes and skeletal variation in patients with malocclusion. Lateral cephalometric radiographs of 269 untreated adults with skeletal classes I, II, and III malocclusion were digitized with 14 landmarks. Two-dimensional coordinates were analyzed using Procrustes fit and principal component (PC) analysis to generate continuous malocclusion phenotypes. Skeletal class classifications (I, II, or III) were used as a categorical phenotype. Individuals were genotyped for 198 single-nucleotide polymorphisms (SNPs) in 71 craniofacial genes and loci. Phenotype-genotype associations were tested via multivariate linear regression for continuous phenotypes and multinomial logistic regression for skeletal malocclusion class. PC analysis resulted in 4 principal components (PCs) explaining 69% of the total skeletal facial variation. PC1 explained 32.7% of the variation and depicted vertical discrepancies ranging from skeletal deep to open bites. PC1 was associated with a SNP near PAX5 (P = 0.01). PC2 explained 21.7% and captured horizontal maxillomandibular discrepancies. PC2 was associated with SNPs upstream of SNAI3 (P = 0.0002) and MYO1H (P = 0.006). PC3 explained 8.2% and captured variation in ramus height, body length, and anterior cranial base orientation. PC3 was associated with TWIST1 (P = 0.000076). Finally, PC4 explained 6.6% and detected variation in condylar inclination as well as symphysis projection. PC4 was associated with PAX7 (P = 0.007). Furthermore, skeletal class II risk increased relative to class I with the minor alleles of SNPs in FGFR2 (odds ratio [OR] = 2.1, P = 0.004) and declined with SNPs in EDN1 (OR = 0.5, P = 0.007). Conversely, skeletal class III risk increased versus class I with SNPs in FGFR2 (OR 2.2, P = 0.005) and COL1A1 (OR = 2.1, P = 0.008) and declined with SNPs in TBX5 (OR = 0.5, P = 0.014). PAX5, SNAI3, MYO1H, TWIST1, and PAX7 are associated with craniofacial skeletal variation among patients with malocclusion, while FGFR2, EDN1, TBX5, and COL1A1 are associated with type of skeletal malocclusion.

An active retrotransposon in Candida albicans.

Tca2 is a Ty1/copia-type retrotransposon from the pathogenic yeast Candida albicans. It was originally identified as an abundant, linear, extrachromosomal, double-stranded DNA molecule. Here we show that Tca2 is widespread in C.albicans, but that the abundance of extrachromosomal Tca2 DNA varies greatly among different strains and is strongly dependent on the growth temperature. The relative levels of Tca2 RNA vary in a similar pattern to the extrachromosomal DNA, raising the possibility that the variations in extrachromosomal DNA levels are introduced predominantly at the level of transcription. We have also analysed the retrotranspositional activity of the element by developing a transposition assay involving a cloned Tca2 element tagged with a selectable marker gene that is activated by passage through an RNA intermediate. We show that the marked Tca2 is transpositionally active as transposed copies of the marked element became integrated at a variety of new positions in the genome and an intron in the donor element was precisely removed in the newly transposed copies. This is the first report of transposition in C.albicans.