A small cohort of FRUM and Engrailed-expressing neurons mediate successful copulation in Drosophila melanogaster.

BMC Neurosci. 2013 May 21;14(1):57

Authors: Latham KL, Liu YS, Taylor BJ

Abstract
BACKGROUND: In Drosophila, male flies require the expression of the male-specific Fruitless protein (FRUM) within the developing pupal and adult nervous system in order to produce male courtship and copulation behaviors. Recent evidence has shown that specific subsets of FRUM neurons are necessary for particular steps of courtship and copulation. In these neurons, FRUM function has been shown to be important for determining sex-specific neuronal characteristics, such as neurotransmitter profile and morphology. RESULTS: We identified a small cohort of FRUM interneurons in the brain and ventral nerve cord by their co-expression with the transcription factor Engrailed (En). We used an En-GAL4 driver to express a fruM RNAi construct in order to selectively deplete FRUM in these En/FRUM co-expressing neurons. In courtship and copulation tests, these males performed male courtship at wild-type levels but were frequently sterile. Sterility was a behavioral phenotype as these En-fruMRNAi males were less able to convert a copulation attempt into a stable copulation, or did not maintain copulation for long enough to transfer sperm and/or seminal fluid. CONCLUSIONS: We have identified a population of interneurons necessary for successful copulation in Drosophila. These data confirm a model in which subsets of FRUM neurons participate in independent neuronal circuits necessary for individual steps of male behavior. In addition, we have determined that these neurons in wild-type males have homologues in females and fru mutants, with similar placement, projection patterns, and neurochemical profiles.

PMID: 23688386 [PubMed - as supplied by publisher]

Inhibiting cancer metastasis via targeting NAPDH oxidase 4.

Biochem Pharmacol. 2013 May 17;

Authors: Zhang B, Liu Z, Hu X

Abstract
Cancer metastasis is a major cause for cancer-related death and inhibiting cancer metastasis is an alternative way to treat cancer. Several lines of reported evidence suggest that NADPH oxidase 4 (NOX4) is a potential target for intervention of cancer metastasis, as the reactive oxygen species (ROS) generated by this enzyme plays important roles in TGF-β signaling, an important inducer of cancer metastasis. Here we show (1) that TGF-β induces ROS production in breast cancer 4T1 cells and enhances cell migration and that the effect of TGF-β depends on NOX4 expression, (2) that knockdown of NOX4 via RNAi significantly decreases the migration ability of 4T1 cells in the presence or absence of TGF-β and significantly attenuates distant metastasis of 4T1 cells to lung and bone, (3) that Schisandrin B (Sch B), a naturally-occurring dibenzocyclooctadiene lignan with very low toxicity, is a novel NOX inhibitor and its IC50 toward NOX4 is 9.3μM, and (4) that Sch B suppresses TGF-β-induced and NOX4-associated ROS production in 4T1 cells and inhibits TGF-β-enhanced cell migration. Similar to NOX4 knockdown observed in this study, Sch B significantly attenuated 4T1 cells distant metastasis to lung and bone in our recently-published study. In line with previous reports, the study suggests that pharmacologically targeting NOX4 may be a potential approach to disrupt cancer metastasis.

PMID: 23688500 [PubMed - as supplied by publisher]

[Expression of long noncoding RNA MALAT1 gene in human nasopharyngeal carcinoma cell lines and its biological significance.]

Nan Fang Yi Ke Da Xue Xue Bao. 2013 May 20;33(5):692-697

Authors: Xie LY, Hu ZY, Wang XY, Li ZG

Abstract
OBJECTIVE: To explore the expression of MALAT 1 in nasopharyngeal carcinoma (NPC) and its biological function. METHOD: Real-time PCR was used to detect the expression of MALAT1 in 5-8F, C666-1, CNE-1, CNE-2, HONE-1, 6-10B and NP69 cell lines. CNE-1 cells engineered with MALAT1 RNA interference (RNAi) and RNA activation (RNAa) techniques were examined for changes in cell proliferation, invasion and metastasis using CCK8 assay, colony formation assay, Transwell in vitro invasion assay and wound-healing assay ability. RESULTS: MALAT1 was highly expressed in 5-8F cells with a high metastatic potential, and lowly expressed in normal nasopharyngeal epithelium cells. Overexpression of MALAT1 by RNAa suppressed the expression of E-cadherin, promoted the expression of vimentin and enhanced the proliferation, invasion, and metastasis of CNE-1 cells. CONCLUSION: MALAT1 can enhance the proliferation, invasion, and metastasis of CNE-1 cells.

PMID: 23688988 [PubMed - as supplied by publisher]

Smad3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway.

Reproduction. 2013 May 20;

Authors: Xu J, Li Y, Yujie J, Liu Y, Shen C, Dong J

Abstract
The function of Smad3, a downstream signaling protein of TGFβ pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells in rat were studied. Female rats (21d SD) were intraperitoneally injected with PMSG 20IU, and granulosa cells were harvested for primary culture 48 hours later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of PCNA (proliferating cell nuclear antigen), cyclin D2, TGFβR II (transforming growth factor-β receptor II), PKA (protein kinase A), FSHR (follicle stimulating hormone receptor) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by enzyme-linked immunosorbent assay (ELISA). Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of granulosa cells. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation, and increased apoptosis of granulosa cells. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.

PMID: 23690627 [PubMed - as supplied by publisher]

Macrophage Migration and Invasion Is Regulated by MMP10 Expression.

PLoS One. 2013;8(5):e63555

Authors: Murray MY, Birkland TP, Howe JD, Rowan AD, Fidock M, Parks WC, Gavrilovic J

Abstract
This study was designed to identify metalloproteinase determinants of macrophage migration and led to the specific hypothesis that matrix metalloproteinase 10 (MMP10/stromelysin-2) facilitates macrophage migration. We first profiled expression of all MMPs in LPS-stimulated primary murine bone marrow-derived macrophages and Raw264.7 cells and found that MMP10 was stimulated early (3 h) and down-regulated later (24 h). Based on this pattern of expression, we speculated that MMP10 plays a role in macrophage responses, such as migration. Indeed, using time lapse microscopy, we found that RNAi silencing of MMP10 in primary macrophages resulted in markedly reduced migration, which was reversed with exogenous active MMP10 protein. Mmp10 (-/-) bone marrow-derived macrophages displayed significantly reduced migration over a two-dimensional fibronectin matrix. Invasion of primary wild-type macrophages into Matrigel supplemented with fibronectin was also markedly impaired in Mmp10 (-/-) cells. MMP10 expression in macrophages thus emerges as an important moderator of cell migration and invasion. These findings support the hypothesis that MMP10 promotes macrophage movement and may have implications in understanding the control of macrophages in several pathologies, including the abnormal wound healing response associated with pro-inflammatory conditions.

PMID: 23691065 [PubMed - as supplied by publisher]

N-Glycosylation Is Required for Secretion and Mitosis in C. elegans.

PLoS One. 2013;8(5):e63687

Authors: Stevens J, Spang A

Abstract
N-glycosylation of proteins is an essential process, and N-glucans serve as important beacons in protein folding and ER associated degradation. More importantly, N-glycosylation increases the structural repertoire of proteins because the addition of the N-glucan on proteins will serve as a base for further sugar additions in the Golgi apparatus, and hence complex three-dimensional structures can be build. N-glycosylation is mediated by the ER-resident OST complex, which is essential throughout eukaryotes. Partial knockdown of conserved OST complex members, such as C. elegans RIBO-1, led to an embryonic lethal phenotype. Although the ER morphology was not grossly altered in ribo-1(RNAi) oocytes and embryos, secretion of yolk and of the yolk receptor RME-2 was perturbed in those worms. Perhaps as a consequence of reduced arrival of N-glycosylated proteins at the plasma membrane, cytokinesis occurred less efficiently leading to multinuclear cells. Unexpectedly, we detected a chromosome segregation defect in ribo-1(RNAi) embryos suggesting an essential role of at least one N-glycosylated protein in metaphase-anaphase transition.

PMID: 23691084 [PubMed - as supplied by publisher]

Potent Inhibition of Hendra Virus Infection via RNA Interference and Poly I:C Immune Activation.

PLoS One. 2013;8(5):e64360

Authors: McCaskill JL, Marsh GA, Monaghan P, Wang LF, Doran T, McMillan NA

Abstract
Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi) based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs) targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

PMID: 23691205 [PubMed - as supplied by publisher]

Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei.

Biocell. 2012 Dec;36(3):133-41

Authors: Zukas R, Chang AJ, Rice M, Springer AL

Abstract
Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Flagellar function in this organism is critical for life cycle progression and pathogenesis, however the regulation of flagellar motility is not well understood. The flagellar axoneme produces a complex beat through the precisely coordinated firing of many proteins, including multiple dynein motors. These motors are found in the inner arm and outer arm complexes. We are studying one of the inner arm dynein motors in the T. brucei flagellum: dynein-f. RNAi knockdown of genes for two components of dynein-f: DNAH10, the alpha heavy chain, and IC138, an intermediate chain, cause severe motility defects including immotility. To determine if motility defects result from structural disruption of the axoneme, we used two different flagellar preparations to carefully examine axoneme structure in these strains using transmission electron microscopy (TEM). Our analysis showed that inner arm dynein size, axoneme structural integrity and fixed central pair orientation are not significantly different in either knockdown culture when compared to control cultures. These results support the idea that immotility in knockdowns affecting DNAH10 or IC138 results from loss of dynein-f function rather than from obvious structural defects in the axoneme.

PMID: 23682429 [PubMed - in process]

The C. elegans gene pan-1 encodes novel transmembrane and cytoplasmic leucine-rich repeat proteins and promotes molting and the larva to adult transition.

BMC Dev Biol. 2013 May 17;13(1):21

Authors: Gissendanner CR, Kelley TD

Abstract
BACKGROUND: Extracellular leucine-rich repeat (eLRR) proteins are a highly diverse superfamily of membrane-associated or secreted proteins. In the membrane-associated eLRR proteins, the leucine-rich repeat motifs interact with the extracellular matrix and other ligands. Characterizing their functions in animal model systems is key to deciphering their activities in various developmental processes. RESULTS: In this study, we identify pan-1 as a critical regulator of C. elegans larval development. pan-1 encodes both transmembrane and cytoplasmic isoforms that vary in the presence and number of leucine-rich repeats. RNAi experiments reveal that pan-1 is required for developmental processes that occur during the mid to late larval stages. Specifically, pan-1 loss of function causes a late larval arrest with a failure to complete development of the gonad, vulva, and hypodermis. pan-1 is also required for early larval ecdysis and execution of the molting cycle at the adult molt. We also provide evidence that pan-1 functionally interacts with the heterochronic gene lin-29 during the molting process. CONCLUSIONS: We show that PAN-1 is a critical regulator of larval development. Our data suggests that PAN-1 promotes developmental progression of multiple tissues during the transition from a larva to a reproductive adult. We further demonstrate that the activity of PAN-1 is complex with diverse roles in the regulation of animal development.

PMID: 23682709 [PubMed - as supplied by publisher]

siRNAs modified with boron cluster and their physicochemical and biological characterization.

Bioconjug Chem. 2013 May 17;

Authors: Lesnikowski ZJ, Olejniczak A, Janczak S, Nawrot B, Sobczak M, Mikolajczyk B, Kwiatkowska A, Sochacki M

Abstract
RNA interference (RNAi) technology provides a powerful, yet selective molecular tool to reduce an expression of genes in eukaryotic cells. Despite of the success associated with the effective use of siRNA duplexes for gene silencing, there is a need to improve their properties. These properties, related mainly to migration through the cell membranes, stability of siRNA in vivo and specificity of their silencing activity, can be improved by chemical modifications of siRNA backbone. In this study, we examined the physicochemical and biological properties of siRNA duplexes targeted against BACE1 gene modified at various positions with a lipophilic boron cluster (C2B10H11, CB). The lipophilicity and resistance to enzymatic degradation of the modified oligomers was higher than the unmodified counterparts. As measured in a dual fluorescence assay (BACE1-GFP/RFP), the carboranyl siRNAs (CB-siRNAs) occurred to be as active as the parent non-modified duplexes and their toxicity towards HeLa cells was also similar. The helical structure of CB-siRNAs remained unchanged upon boron cluster introduction, as determined by CD and UV melting experiments.

PMID: 23682800 [PubMed - as supplied by publisher]