Zebrafish larvae are ideally suited for high-throughput analyses of vertebrate behavior. The larvae can be examined in multiwell plates and display a range of behaviors during early development. Previous studies have shown that zebrafish larvae display a preference for the edge of the well and several lines of evidence suggest this edge preference (thigmotaxis) may be a measure of anxiety. In the present study, we further examined the relation between edge preference and anxiety by imaging zebrafish larvae exposed to three psychoactive drugs diazepam (Valium), fluoxetine (Prozac), and caffeine. The edge preference was first examined in a five-fish assay, with and without visual stimuli. Diazepam, a benzodiazepine that binds to GABA receptors, reduced the larval edge preference, with or without visual stimuli. In contrast, fluoxetine, a selective serotonin reuptake inhibitor, did not affect the edge preference. Caffeine increased the preference for the edge in response to visual stimuli. Similar effects were observed in a two-fish assay; diazepam-exposed larvae showed a reduced edge preference and caffeine exposed larvae showed an increased edge preference. These results suggest that the edge preference in zebrafish larvae is a measure of anxiety and further illustrate that the pharmaceuticals used in the study have different mechanisms of action. Although there are substantial differences between zebrafish and human brains, our results indicate that the signals that regulate anxiety are similar on a molecular level. We propose that high-throughput assays in zebrafish may be used to uncover genetic or environmental factors that cause anxiety disorders and may contribute to the development of novel strategies to prevent or treat such disorders.

Striatal neuropathology of Huntington's disease (HD) involves primary and progressive degeneration of the medium-sized projection neurons, with relative sparing of the local circuit interneurons. The mechanism for such a patterned cell loss in the HD striatum continues to remain unclear. Optineurin (OPTN) is one of the proteins interacting with huntingtin and plays a protective role in several neurodegenerative disorders. To determine the cellular localization pattern of OPTN in the mouse striatum, we employed a highly sensitive immunohistochemistry with the tyramide signal amplification system. In this study, we show that OPTN appeared as a cytoplasmic protein within the subsets of the striatal neurons. Of particular interest was that OPTN was abundantly expressed in the interneurons, whereas low levels of OPTN were observed in the medium projection neurons. This cell type-specific distribution of OPTN in the striatum is strikingly complementary to the pattern of neuronal loss typically observed in the striatum of patients with HD. We suggest that OPTN abundance is an important cellular factor in considering the cell type-specific vulnerability of striatal neurons in HD.

Both histone-acetylations and histone deacetylases have been shown to play a key role in cardiac remodeling. Recently, it has become abundantly clear that many non-histone proteins are modified by post-translational lysine acetylations and that these acetylations regulate protein activity, conformation, and binding. In the present study, non-histone acetylated proteins associated with heart failure were identified. Global screening for lysine acetylated proteins was performed using 2-dimensional gel electrophoresis coupled with immunoblotting with a primary monoclonal anti-acetyl-lysine antibody. Lysine acetylated proteins were compared in two rodent models of hypertensive heart failure, the Dahl salt-sensitive (SS) and spontaneously hypertensive heart failure prone (SHHF) rats with those in corresponding controls, i.e., the Dahl salt-resistant (SR) and W (W) rat strains, respectively. Forty-one and 66 acetylated proteins were detected in SS and SHHF failing hearts, respectively, but either not detected or detected with less abundance in corresponding control hearts. Twelve of these acetylated proteins were common to both models of heart failure. These were identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry followed by Mascot Analysis and included mitochondrial enzymes: ATP synthase, long-chain acyl-CoA dehydrogenase, creatine kinase, malate dehydrogenase, and pyruvate dehydrogenase. The abundance of NAD-dependent deacetylase sirtuin-3 (Sirt3), a mitochondrial deacetylase was reduced in SS and SHHF failing hearts. This is the first description of non-histone protein acetylations associated with heart failure and raises the prospect that acetylations of mitochondrial proteins linked to reduced Sirt3 mediate, in part, metabolic changes in heart failure.

Wnt signaling is critical for skeletal development and homeostasis. Sclerostin (Sost) has emerged as a potent inhibitor of Wnt signaling and, thereby, bone formation. Thus, strategies to reduce sclerostin expression may be used to treat osteoporosis or non-union fractures. Transforming growth factor-beta (TGF-β) elicits various effects upon the skeleton both in vitro and in vivo depending on the duration and timing of administration. In vitro and in vivo studies demonstrate that TGF-β increases osteoprogenitor differentiation but decreases matrix mineralization of committed osteoblasts. Because sclerostin decreases matrix mineralization, this study aimed to examine whether TGF-β achieves such inhibitory effects via transcriptional modulation of Sost. Using the UMR106.01 mature osteoblast cell line, we demonstrated that TGF-βTGF-β(1)-β(2)-β(3) and Activin A increase Sost transcript expression. Pharmacologic inhibition of Alk4/5/7 in vitro and in vivo decreased endogenous Sost expression, and siRNA against Alk4 and Alk5 demonstrated their requirement for endogenous Sost expression. TGF-β(1) targeted the Sost bone enhancer ECR5 and did not affect the transcriptional activity of the endogenous Sost promoter. These results indicate that TGF-β(1) controls Sost transcription in mature osteoblasts, suggesting that sclerostin may mediate the inhibitory effect of TGF-β upon osteoblast differentiation.

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and can serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. miRNAs as well as a large population of putative piRNAs (piwi-interacting RNAs) had dynamic accumulation profiles through early development. Defects in morphogenesis caused by loss of Drosha could be rescued with four miRNAs. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. Adhesion on U937 cells, expression of adhesion molecules, production of ROS and NO, expression of eNOS, and translocation of NF-κB were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, or both. High concentrations of glucose (>20mM) induced an increase in adhesion, an increment in mainly E-selectin and PECAM-1 expression, as well as in ROS and NO production, eNOS expression, translocation of NF-κB, and degradation of its inhibitor IκB-α. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative stress and decreased translocation of NF-κB and the degradation of IκB-α induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes.

Since data-mining from the Oncomine database revealed that expression of histone H2B K120 monoubiquitin (H2Bub1) ligase RNF20 is decreased in metastatic prostate cancer, we elucidated the effect of RNF20 and its homolog RNF40 on androgen receptor (AR)-dependent transcription and prostate cancer cell growth. Both RNF20 and RNF40 were able to functionally and physically interact with the AR and modulate its transcriptional activity in intact cells. Chromatin immunoprecipitation analyses showed that the androgen induction of FKBP51 and PSA in LNCaP prostate cancer cells is accompanied with a dynamic increase in the H2Bub1 within the transcribed regions of these loci. Interestingly, depletion of RNF20 or RNF40 strongly retarded the growth of LNCaP cells, which was however unlikely to be due to altered androgen signaling, but due to decreased expression of several cell cycle promoters. Collectively, our results suggest that RNF20 and RNF40, either via ubiquitylation of H2B or other targets, are coupled to the proliferation of prostate cancer cells.

Here we report on the application of cationic liposomes formed by new cationic lipids and the lipid-helper DOPE (dioleoylphosphatidylethanolamine) for the transfection of plasmid DNA and mRNA into dendritic cells (DCs) progenitors and immature DCs of bone-marrow origin in vitro and the use of these DCs to induce the suppression of B16 melanoma metastases in vivo. The cationic lipids contain one (X2, S1, S2 and S3) or two (2X3) cholesterol residues or long-chain hydrocarbon substituent (2D3) linked with spermine. Data show that liposomes 2X3-DOPE, 2D3-DOPE, X2-DOPE and S2-DOPE display high transfection efficiency in respect to DNA (30-47% of DC progenitors and up to 57% of immature DC were transfected) and RNA (up to 57% of cells were transfected). The studied lipids exhibited an efficiency of DNA and RNA delivery in DCs several times higher in comparison with Lipofectamine 2000. Observed ex vivo the higher transfection efficiencies of DCs with mRNAs encoding of a set of tumor-associated antigens provided by cationic liposomes 2X3-DOPE and 2X2-DOPE corresponded to a 3-5 fold suppression of metastasis number in a model of murine B16 melanoma in vivo. The injection into mice of these pulsed DCs resulted in a slight pro-inflammatory response which was balanced by the positive effect of the antitumor cytokine production induced by the DCs. The obtained data show that the novel spermine-based polycationic lipids can be applied in the preparation of antitumor DC-based vaccine.

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system.

G protein-coupled receptor 17 (GPR17), the new P2Y-like receptor, is phylogenetically related to the P2Y and cysteinyl leukotriene receptors, and responds to both uracil nucleotides and cysteinyl leukotrienes. GPR17 has been proposed to be a damage sensor in ischemic stroke; however, its role in brain inflammation needs further detailed investigation. Here, we extended previous studies on the spatiotemporal profiles of GPR17 expression and localization, and their implications for brain injury after focal cerebral ischemia. We found that in the ischemic core, GPR17 mRNA and protein levels were upregulated at both 12-24 h and 7-14 days, but in the boundary zone the levels increased 7-14 days after reperfusion. The spatiotemporal pattern of GPR17 expression well matched the acute and late (subacute/chronic) responses in the ischemic brain. According to previous findings, in the acute phase, after ischemia (24 h), upregulated GPR17 was localized in injured neurons in the ischemic core and in a few microglia in the ischemic core and boundary zone. In the late phase (14 days), it was localized in microglia, especially in activated (ED1-positive) microglia in the ischemic core, but weakly in most microglia in the boundary zone. No GPR17 was detectable in astrocytes. GPR17 knockdown by a small interfering RNA attenuated the neurological dysfunction, infarction, and neuron loss at 24 h, and brain atrophy, neuron loss, and microglial activation at 14 days after reperfusion. Thus, GPR17 might mediate acute neuronal injury and late microgliosis after focal cerebral ischemia.

Changes in gene expression during development play an important role in shaping morphological and behavioral differences, including between humans and nonhuman primates. Although many of the most striking developmental changes occur during early development, reproductive maturation represents another critical window in primate life history. However, this process is difficult to study at the molecular level in natural primate populations. Here, we took advantage of ovarian samples made available through an unusual episode of human-wildlife conflict to identify genes that are important in this process. Specifically, we used RNA sequencing (RNA-Seq) to compare genome-wide gene expression patterns in the ovarian tissue of juvenile and adult female baboons from Amboseli National Park, Kenya. We combined this information with prior evidence of selection occurring on two primate lineages (human and chimpanzee). We found that in cases in which genes were both differentially expressed over the course of ovarian maturation and also linked to lineage-specific selection this selective signature was much more likely to occur in regulatory regions than in coding regions. These results suggest that adaptive change in the development of the primate ovary may be largely driven at the mechanistic level by selection on gene regulation, potentially in relationship to the physiology or timing of female reproductive maturation.

A family of tissue-specific auxiliary β subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, β subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying β subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a β1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of β1. These amino acids reside within two regions of highest conservation among related β1, β2, and β4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the β1 Y74 and S104-I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for β subunit modulation of voltage-gated potassium channels wherein interactions between extracellular β subunit residues with the external portions of the gate and voltage sensor regulate channel opening.

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.

A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ribosome association on the ER. Here, we provide evidence that p180 is required to form translationally active polysome/translocon complexes on the ER. Assembly of highly-developed polysomes on the ER was severely perturbed upon loss of p180. p180 associates with polysome/translocon complexes through multiple contact sites: it was coimmunoprecipitated with the translocon complex independently of ribosomes, while it can also bind to ribosomal large subunit specifically. The responsible domain of p180 for membrane polysome assembly was identified in the C-terminal coiled-coil region. The degree of ribosome occupation of collagen and fibronectin mRNAs was regulated in response to increased traffic demands. This effect appears to be exerted in a manner specific for a specified set of mRNAs. Collectively, our data suggest that p180 is required to form translationally active polysome/translocon complexes on the ER membrane, and plays a pivotal role in highly efficient biosynthesis on the ER membrane through facilitating polysome formation in professional secretory cells.

Predicting the location and strength of promoters from genomic sequence requires accurate sequenced-based promoter models. We present the first model of a full-length bacterial promoter, encompassing both upstream sequences (UP-elements) and core promoter modules, based on a set of 60 promoters dependent on σ(E), an alternative ECF-type σ factor. UP-element contribution, best described by the length and frequency of A- and T-tracts, in combination with a PWM-based core promoter model, accurately predicted promoter strength both in vivo and in vitro. This model also distinguished active from weak/inactive promoters. Systematic examination of promoter strength as a function of RNA polymerase (RNAP) concentration revealed that UP-element contribution varied with RNAP availability and that the σ(E) regulon is comprised of two promoter types, one of which is active only at high concentrations of RNAP. Distinct promoter types may be a general mechanism for increasing the regulatory capacity of the ECF group of alternative σ's. Our findings provide important insights into the sequence requirements for the strength and function of full-length promoters and establish guidelines for promoter prediction and for forward engineering promoters of specific strengths.

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site.

Tumour necrosis factor (TNF) is upregulated in the alveolar space early in the course of ventilator-induced lung injury (VILI). Studies in genetically modified mice indicate that the two TNF receptors play opposing roles during injurious high-stretch mechanical ventilation, with p55 promoting but p75 preventing pulmonary oedema.

Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

Preoperative characterization of thyroid follicular lesions is challenging. Fine-needle aspiration specimens cannot differentiate follicular carcinomas from benign follicular neoplasias. Recently, promising markers have been detected using modern molecular techniques. We conducted a retrospective study to confirm the usefulness of immunohistochemical staining for the protein markers, DDIT3, STT3A (ITM1), ARG2 and FAM129A (C1orf24) in separating benign and malignant thyroid follicular lesions. Formalin-fixed, paraffin-embedded thyroid tissue from 30 in-house cases (15 follicular carcinomas and 15 follicular adenomas), as well as 8 follicular carcinomas and 21 follicular adenomas on tissue microarray slides were stained immunohistochemically for DDIT3, STT3A, ARG2 and FAM129A expression. Control tissue consisted of thyroid parenchyma adjacent to the tumors and 11 separate cases of normal thyroid parenchyma. All in-house cases of follicular adenomas, follicular carcinomas and adjacent normal thyroid tissue showed positive immunostaining with anti-DDIT3 and anti-STT3A. Anti-ARG2 and anti-FAM129A polyclonal antibodies showed positive staining in 20 and 60% of in-house follicular adenomas, and 40 and 87% of in-house follicular carcinomas, respectively. Monoclonal anti-FAM129A demonstrated positive staining in 13 and 33% of in-house follicular adenomas and follicular carcinomas, respectively. Polyclonal anti-DDIT3, -STT3A and -FAM129A antibodies showed positive staining in all tissue microarray slides of follicular carcinoma and in 76, 85 and 81% of the follicular adenomas, respectively. Monoclonal anti-STT3A stained 81% of the follicular adenoma cores. Anti-ARG2 stained positive in 13% of follicular carcinomas and 10% of follicular adenomas on the tissue microarray slides. In conclusion, DDIT3, STT3A, ARG2 and FAM129A immunohistochemistry does not appear to be useful in the diagnosis of thyroid follicular neoplasias, as they do not reliably distinguish follicular thyroid carcinoma from follicular thyroid adenoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600 000 new cases diagnosed each year. Understanding the molecular pathways that lead to HNSCC is crucial to identify new targets for anti-cancer drug development. Protein kinase Cɛ (PKCɛ) is elevated in HNSCC and regulates the activation of Akt, Stat3 and Rho GTPases. To date, the molecular mechanism of PKCɛ dysregulation in HNSCC remains to be elucidated. In silico analysis identified three putative microRNA-107 (miR-107) binding sites in the 3'-untranslated region (UTR) of PKCɛ. An inverse relationship was revealed between miR-107 and PKCɛ in HNSCC cell lines. Delivery of miR-107 reduced PKCɛ levels in SCC15, SCC25 and CAL27, three HNSCC cell lines with high PKCɛ and low miR-107. The activity of a luciferase reporter construct containing the 3'-UTR of PKCɛ was downregulated by miR-107 and mutations in the three cognate miR-107 binding sites completely ablated the regulation by miR-107. Treatment with miR-107 significantly blocked cell proliferation, DNA replication, colony formation and invasion in SCC25 and CAL27 cells. Ectopic expression of miR-resistant PKCɛ was sufficient to partially rescue the loss-of-function phenotype in miR-107-overexpressing SCC25 cells. Tumor growth in nude mice was retarded by 93±7% in CAL27/miR-107 cells compared with CAL27/miR-control cells. Last, human primary HNSCC tumors with elevated PKCɛ had reduced miR-107 expression. Our results demonstrate that PKCɛ is directly regulated by miR-107 and, moreover, suggest that miR-107 may be a potential anti-cancer therapeutic for HNSCC.