sPARTA: a parallelized pipeline for integrated analysis of plant miRNA and cleaved mRNA data sets,including new miRNA target-identification software

Parallel analysis of RNA ends (PARE) is a technique utilizing high-throughput sequencing to profile uncapped, mRNA cleavage or decay products on a genome-wide basis. Tools currently available to validate miRNA targets using PARE data employ only annotated genes, whereas important targets may be found in unannotated genomic regions. To handle such cases and to scale to the growing availability of PARE data and genomes, we developed a new tool, ‘sPARTA’ (small RNA-PARE target analyzer) that utilizes a built-in, plant-focused target prediction module (aka ‘miRferno’). sPARTA not only exhibits an unprecedented gain in speed but also it shows greater predictive power by validating more targets, compared to a popular alternative. In addition, the novel ‘seed-free’ mode, optimized to find targets irrespective of complementarity in the seed-region, identifies novel intergenic targets. To fully capitalize on the novelty and strengths of sPARTA, we developed a web resource, ‘comPARE’, for plant miRNA target analysis; this facilitates the systematic identification and analysis of miRNA-target interactions across multiple species, integrated with visualization tools. This collation of high-throughput small RNA and PARE datasets from different genomes further facilitates re-evaluation of existing miRNA annotations, resulting in a ‘cleaner’ set of microRNAs.     

A mutation in the Cdon gene potentiates congenital nevus development mediated by NRASQ61K

Congenital nevi develop before birth and sometimes cover large areas of the body. They are presumed to arise from the acquisition of a gene mutation in an embryonic melanocyte that becomes trapped in the dermis during development. Mice bearing the Cdk4^R24C::Tyr‐NRAS^Q61K transgenes develop congenital nevus‐like lesions by post‐natal day 10, from melanocytes escaping the confines of hair follicles. We interbred these mice with the collaborative cross (CC), a resource that enables identification of modifier genes for complex diseases (those where multiple genes are involved). We examined variation in nevus cell density in 66 CC strains and mapped a large‐effect quantitative trait locus (QTL) controlling nevus cell density to murine chromosome 9. The best candidate for a gene that exacerbates congenital nevus development in the context of an NRAS mutation is Cdon, a positive regulator of sonic hedgehog (Shh) that is expressed mainly in keratinocytes.     

Vibrio coralliilyticus Strain OCN008 Is an Etiological Agent of Acute Montipora White Syndrome

Identification of a pathogen is a critical first step in the epidemiology and subsequent management of a disease. A limited number of pathogens have been identified for diseases contributing to the global decline of coral populations. Here we describe Vibrio coralliilyticus strain OCN008, which induces acute Montipora white syndrome (aMWS), a tissue loss disease responsible for substantial mortality of the coral Montipora capitata in Kāne‘ohe Bay, Hawai‘i. OCN008 was grown in pure culture, recreated signs of disease in experimentally infected corals, and could be recovered after infection. In addition, strains similar to OCN008 were isolated from diseased coral from the field but not from healthy M. capitata. OCN008 repeatedly induced the loss of healthy M. capitata tissue from fragments under laboratory conditions with a minimum infectious dose of between 10⁷ and 10⁸ CFU/ml of water. In contrast, Porites compressa was not infected by OCN008, indicating the host specificity of the pathogen. A decrease in water temperature from 27 to 23°C affected the time to disease onset, but the risk of infection was not significantly reduced. Temperature-dependent bleaching, which has been observed with the V. coralliilyticus type strain BAA-450, was not observed during infection with OCN008. A comparison of the OCN008 genome to the genomes of pathogenic V. coralliilyticus strains BAA-450 and P1 revealed similar virulence-associated genes and quorum-sensing systems. Despite this genetic similarity, infections of M. capitata by OCN008 do not follow the paradigm for V. coralliilyticus infections established by the type strain.     

Immune enhancing effect of a growth hormone secretagogue

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.     

SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling

The use of small-molecule inhibitors to study molecular components of cellular signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interfering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. A related inhibitor, SU6657, inhibits many kinases, including Src and the platelet-derived growth factor (PDGF) receptor. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. By comparing PDGF-stimulated tyrosine phosphorylation events in untreated and SU6656-treated cells, we found that some substrates (for example, c-Cbl, and protein kinase C delta) were Src family substrates whereas others (for example, phospholipase C-gamma) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases (on tyrosines 239 and 240) and a distinct tyrosine kinase (on tyrosine 317, which is perhaps phosphorylated by the PDGF receptor itself). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain mutation, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosphotyrosine-binding domain also inhibited synthesis. These inhibitions were overcome by heterologous expression of Myc, supporting the hypothesis that Shc functions in the Src pathway. SU6656 should prove a useful additional tool for further dissecting the role of Src kinases in this and other signal transduction pathways.     

Epithelial augmentation of trachealis contraction caused by major basic protein of eosinophils

We studied the effect of epithelial removal and intraepithelial administration of human eosinophil granule major basic protein (MBP) on the contraction of underlying canine tracheal smooth muscle in 23 dogs in vivo. A dual in situ tracheal preparation was utilized that allowed sharp excision of epithelium. The response to intra-arterial acetylcholine (ACh) was augmented substantially in five dogs receiving 200 micrograms MBP by intraepithelial instillation. Active tension elicited by 10(-8) mol intra-arterial ACh was 34.0 +/- 2.2 g/cm before and 46.1 +/- 2.6 g/cm 30 min after MBP (P less than 0.002). There was no change in active tension in the control segment in the same dogs after intraepithelial instillation of vehicle only (34.7 +/- 3.2 vs. 34.4 +/- 2.3 g/cm; P = NS). Instillation of MBP directly into the subepithelial tracheal smooth muscle did not alter contraction. To assess whether this augmentation was caused by inhibition of an epithelial-derived relaxant factor, additional studies were performed in nine other dogs in which the epithelium was excised discretely from one of the two tracheal segments. No significant differences in contractile response to ACh or relaxation response to isoproterenol were observed at 2, 15, 30, or 60 min after epithelial excision. We demonstrate that intraepithelial administration of MBP augments the contraction of underlying canine tracheal smooth muscle elicited by ACh. This augmentation is a direct effect of MBP and does not require antagonism of epithelial inhibition.     

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

Nullisomic deletion of the mcf.2 transforming gene in two haemophilia B patients.

The mcf.2 transforming gene sequence has been located to the region between 29 and 61 kb 3' of the factor IX gene. Two unrelated haemophilia B patients who raise antibodies to infused factor IX ('inhibitors') have deletions in excess of 273 kb encompassing the factor IX and mcf.2 genes and a CG-rich island. We believe these patients show the first nullisomic deletion of a transforming gene to be reported. No clinical condition can be attributed to the loss of the mcf.2 gene.