MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.     

Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly

In mammals, heterochromatin is characterized by DNA methylation at CpG dinucleotides and methylation at lysine 9 of histone H3. It is currently unclear whether there is a coordinated transmission of these two epigenetic modifications through DNA replication. Here we show that the methyl-CpG binding protein MBD1 forms a stable complex with histone H3-K9 methylase SETDB1. Moreover, during DNA replication, MBD1 recruits SETDB1 to the large subunit of chromatin assembly factor CAF-1 to form an S phase-specific CAF-1/MBD1/SETDB1 complex that facilitates methylation of H3-K9 during replication-coupled chromatin assembly. In the absence of MBD1, H3-K9 methylation is lost at multiple genomic loci and results in activation of p53BP2 gene, normally repressed by MBD1 in HeLa cells. Our data suggest a model in which H3-K9 methylation by SETDB1 is dependent on MBD1 and is heritably maintained through DNA replication to support the formation of stable heterochromatin at methylated DNA.     

Molecular characterization and chromosome-specific TRAP-marker development for Langdon durum D-genome disomic substitution lines.

The aneuploid stocks of durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husnot) and common wheat (T. aestivum L.) have been developed mainly in 'Langdon' (LDN) and 'Chinese Spring' (CS) cultivars, respectively. The LDN-CS D-genome chromosome disomic substitution (LDN-DS) lines, where a pair of CS D-genome chromosomes substitute for a corresponding homoeologous A- or B-genome chromosome pair of LDN, have been widely used to determine the chromosomal locations of genes in tetraploid wheat. The LDN-DS lines were originally developed by crossing CS nulli-tetrasomics with LDN, followed by 6 backcrosses with LDN. They have subsequently been improved with 5 additional backcrosses with LDN. The objectives of this study were to characterize a set of the 14 most recent LDN-DS lines and to develop chromosome-specific markers, using the newly developed TRAP (target region amplification polymorphism)-marker technique. A total of 307 polymorphic DNA fragments were amplified from LDN and CS, and 302 of them were assigned to individual chromosomes. Most of the markers (95.5%) were present on a single chromosome as chromosome-specific markers, but 4.5% of the markers mapped to 2 or more chromosomes. The number of markers per chromosome varied, from a low of 10 (chromosomes 1A and 6D) to a high of 24 (chromosome 3A). There was an average of 16.6, 16.6, and 15.9 markers per chromosome assigned to the A-, B-, and D-genome chromosomes, respectively, suggesting that TRAP markers were detected at a nearly equal frequency on the 3 genomes. A comparison of the source of the expressed sequence tags (ESTs), used to derive the fixed primers, with the chromosomal location of markers revealed that 15.5% of the TRAP markers were located on the same chromosomes as the ESTs used to generate the fixed primers. A fixed primer designed from an EST mapped on a chromosome or a homoeologous group amplified at least 1 fragment specific to that chromosome or group, suggesting that the fixed primers might generate markers from target regions. TRAP-marker analysis verified the retention of at least 13 pairs of A- or B-genome chromosomes from LDN and 1 pair of D-genome chromosomes from CS in each of the LDN-DS lines. The chromosome-specific markers developed in this study provide an identity for each of the chromosomes, and they will facilitate molecular and genetic characterization of the individual chromosomes, including genetic mapping and gene identification.     

Validation of Verbal Autopsy Tool for Ascertaining the Causes of Stillbirth

Objective

To assess performance of the WHO revised verbal autopsy tool for ascertaining the causes of still birth in comparison with reference standard cause of death ascertained by standardized clinical and supportive data.

Methods

All stillbirths at a tertiary hospital in Karachi, Pakistan were prospectively recruited into study from August 2006- February 2008. The reference standard cause of death was established by two senior obstetricians within 48 hours using the ICD coding system. Verbal autopsy interviews using modified WHO tool were conducted by trained health workers within 2- 6 weeks of still birth and the cause of death was assigned by second panel of obstetricians. The performance was assessed in terms of sensitivity, specificity and Kappa.

Results

There were 204 still births. Of these, 80.8% of antepartum and 50.5% of intrapartum deaths were correctly diagnosed by verbal autopsy. Sensitivity of verbal autopsy was highest 68.4%, (95%CI: 46-84.6) for congenital malformation followed by obstetric complication 57.6%, (95%CI: 25-84.2). The specificity for all major causes was greater than 90%. The level of agreement was high (kappa=0.72) for anomalies and moderate (k=0.4) for all major causes of still birth, except asphyxia.

Conclusion

Our results suggest that verbal autopsy has reasonable validity in identifying and discriminating between causes of stillbirth in Pakistan. On the basis of these findings, we feel it has a place in resource constrained areas to inform strategic planning and mobilization of resources to attain Millennium Development Goals.     

Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.     

Versican G3 domain promotes blood coagulation through suppressing the activity of tissue factor pathway inhibitor-1

We have detected versican, a member of the large chondroitin sulfate proteoglycans, and its degraded C-terminal G3 fragments in human plasma and observed that the versican G3 domain promoted blood coagulation. Silencing G3 expression with small interfering RNA reduced the effect of G3 on coagulation. Plasma coagulation assays suggest that G3 enhances coagulation irrespective of its actions on platelets and white blood cells. To examine how versican affected blood coagulation, we used normal human plasma and different types of coagulation factor-deficient plasmas. The experiments indicated that versican enhanced coagulation through the extrinsic pathway, and that Factor VII was the target molecule. FVII activity assays showed that G3 activated FVII in the presence of plasma but not with purified FVII directly. Yeast two-hybrid, immunoprecipitation, and gel co-migration assays showed that G3 interacted with the tissue factor pathway inhibitor-1 (TFPI-1). TFPI-1 activity assays suggested that G3 inhibited TFPI-1 activity, allowing FVIIa and FXa to facilitate the coagulation process. G3-induced blood coagulation was further confirmed with a mouse model in a real-time manner. Taken together, these results indicate that versican may represent a new target for the development of therapies against atherosclerosis.