hnRNP L is essential for myogenic differentiation and modulates myotonic dystrophy pathologies.

INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.

Molecular and Functional Properties of Regional Astrocytes in the Adult Brain.

The molecular signature and functional properties of astroglial subtypes in the adult CNS remain largely undefined. By using translational ribosome affinity purification followed by RNA-Seq, we profiled astroglial ribosome-associated (presumably translating) mRNAs in major cortical and subcortical brain regions (cortex, hippocampus, caudate-putamen, nucleus accumbens, thalamus, and hypothalamus) of BAC aldh1l1-translational ribosome affinity purification (TRAP) mice (both sexes). We found that the expression of astroglial translating mRNAs closely follows the dorsoventral axis, especially from cortex/hippocampus to thalamus/hypothalamus posteriorly. This region-specific expression pattern of genes, such as synaptogenic modulator sparc and transcriptional factors (emx2, lhx2, and hopx), was validated by qRT-PCR and immunostaining in brain sections. Interestingly, cortical or subcortical astrocytes selectively promote neurite growth and synaptic activity of neurons only from the same region in mismatched cocultures, exhibiting region-matched astrocyte to neuron communication. Overall, these results generated new molecular signature of astrocyte types in the adult CNS, providing insights into their origin and functional diversity.SIGNIFICANCE STATEMENT We investigated the in vivo molecular and functional heterogeneity of astrocytes inter-regionally from adult brain. Our results showed that the expression pattern of ribosome-associated mRNA profiles in astrocytes closely follows the dorsoventral axis, especially posteriorly from cortex/hippocampus to thalamus/hypothalamus. In line with this, our functional results further demonstrated region-selective roles of cortical and subcortical astrocytes in regulating cortical or subcortical neuronal synaptogenesis and maturation. These in vivo studies provide a previously uncharacterized and important molecular atlas for exploring region-specific astroglial functions.

Molecular analysis of acute and chronic reactive astrocytes in the pilocarpine model of temporal lobe epilepsy.

Astroglia, the most abundant glial cells in the mammalian central nervous system (CNS), are considered an emerging key player in seizure induction and progression. Although astrocytes undergo reactive gliosis in temporal lobe epilepsy (TLE) with dramatic morphological and molecular changes, specific astrocyte targets/molecular pathways that contribute to the induction and progression of seizure remain largely unknown. By combining translating ribosomal affinity purification (TRAP) with the pilocarpine model of TLE in BAC aldh1l1 TRAP mice, we profiled translating mRNAs from hippocampal or cortical astrocytes at different phases (3days, 30days, and 60days post-pilocarpine injections) of pilocarpine-induced epilepsy models. Our results found that hippocampal (but not cortical) astrocytes undergo early and unique molecular changes at 3days post-pilocarpine injections. These changes indicate a potentially primary pathogenic role of hippocampal astrocytes in seizure induction and progression and provide new insights about the involvement of specific astrocytic pathways/targets in epilepsy. In particular, we validated expression changes of ocrl and aeg1 in pilocarpine models. Follow-up studies on these genes may reveal new roles of hippocampal astrocytes in TLE.

One siRNA pool targeting the λ constant region stops λ light-chain production and causes terminal endoplasmic reticulum stress.

In systemic light-chain amyloidosis, λ light chains produced by clonal plasma cells cause organ damage and early death. In pursuit of novel therapy, we developed 1 pool of short interfering RNA (siRNA) targeting the constant region of λ light chains that substantially and promptly reduces λ-light-chain production and secretion by human plasma cells regardless of sequence diversity. In clones producing intact immunoglobulin G (IgG) λ antibodies (containing paired heavy and light chains), the secretion of intact antibodies is reduced, and all 3 branches of the unfolded protein response are activated by accumulation of unpaired IgG heavy chains in the endoplasmic reticulum (ER). Moreover, an ER stress response can then become terminal with effector caspase activity mediated in part by the transcription of the Bcl-2 homology 3 domain only family member NOXA. This pool of siRNA can be used to reduce pathological λ-light-chain production and cause apoptosis in human plasma cells making intact IgGλ antibodies.

Estrogen receptor-mediated regulation of microRNA inhibits proliferation of vascular smooth muscle cells.

OBJECTIVE: Estradiol (E2) regulates gene transcription by activating estrogen receptor-α and estrogen receptor-ß. Many of the genes regulated by E2 via estrogen receptors are repressed, yet the molecular mechanisms that mediate E2-induced gene repression are currently unknown. We hypothesized that E2, acting through estrogen receptors, regulates expression of microRNAs (miRs) leading to repression of expression of specific target genes. METHODS AND RESULTS: Here, we report that E2 significantly upregulates the expression of 26 miRs and downregulates the expression of 6 miRs in mouse aorta. E2-mediated upregulation of one of these miRs, miR-203, was chosen for further study. In cultured vascular smooth muscle cells (VSMC), E2-mediated upregulation of miR-203 is mediated by estrogen receptor-α (but not estrogen receptor-ß) via transcriptional upregulation of the primary miR. We demonstrate that the transcription factors Zeb-1 and AP-1 play critical roles in mediating E2-induced upregulation of miR-203 transcription. We show further that miR-203 mediates E2-induced repression of Abl1, and p63 protein abundance in VSMC. Finally, knocking-down miR-203 abolishes E2-mediated inhibition of VSMC proliferation, and overexpression of miR-203 inhibits cultured VSMC proliferation, but not vascular endothelial cell proliferation. CONCLUSIONS: Our findings demonstrate that E2 regulates expression of miRs in the vasculature and support the estrogen receptors-dependent induction of miRs as a mechanism for E2-mediated gene repression. Furthermore, our findings demonstrate that miR-203 contributes to E2-induced inhibition of VSMC proliferation and highlight the potential of miR-203 as a therapeutic agent in the treatment of proliferative cardiovascular diseases.

A program for real-time surveillance of SARS-CoV-2 genetics.

The COVID-19 pandemic brought forth an urgent need for widespread genomic surveillance for rapid detection and monitoring of emerging SARS-CoV-2 variants. It necessitated design, development, and deployment of a nationwide infrastructure designed for sequestration, consolidation, and characterization of patient samples that disseminates de-identified information to public authorities in tight turnaround times. Here, we describe our development of such an infrastructure, which sequenced 594,832 high coverage SARS-CoV-2 genomes from isolates we collected in the United States (U.S.) from March 13th 2020 to July 3rd 2023. Our sequencing protocol ('Virseq') utilizes wet and dry lab procedures to generate mutation-resistant sequencing of the entire SARS-CoV-2 genome, capturing all major lineages. We also characterize 379 clinically relevant SARS-CoV-2 multi-strain co-infections and ensure robust detection of emerging lineages via simulation. The modular infrastructure, sequencing, and analysis capabilities we describe support the U.S. Centers for Disease Control and Prevention national surveillance program and serve as a model for rapid response to emerging pandemics at a national scale.

Rapid estrogen receptor signaling is essential for the protective effects of estrogen against vascular injury.

BACKGROUND: Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which, when bound to estrogen, can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a rapid nonnuclear signaling cascade. However, the biological significance of this rapid signaling pathway has been unclear. METHODS AND RESULTS: In the present study, we develop a novel transgenic mouse in which rapid signaling is blocked by overexpression of a peptide that prevents ERs from interacting with the scaffold protein striatin (the disrupting peptide mouse). Microarray analysis of ex vivo treated mouse aortas demonstrates that rapid ER signaling plays an important role in estrogen-mediated gene regulatory responses. Disruption of ER-striatin interactions also eliminates the ability of estrogen to stimulate cultured endothelial cell migration and to inhibit cultured vascular smooth muscle cell growth. The importance of these findings is underscored by in vivo experiments demonstrating loss of estrogen-mediated protection against vascular injury in the disrupting peptide mouse after carotid artery wire injury. CONCLUSIONS: Taken together, these results support the concept that rapid, nonnuclear ER signaling contributes to the transcriptional regulatory functions of ER and is essential for many of the vasoprotective effects of estrogen. These findings also identify the rapid ER signaling pathway as a potential target for the development of novel therapeutic agents.

An In Silico Analysis of PCR-Based Monkeypox Virus Detection Assays: A Case Study for Ongoing Clinical Surveillance.

The 2022 global Mpox outbreak swiftly introduced unforeseen diversity in the monkeypox virus (MPXV) population, resulting in numerous Clade IIb sublineages. This propagation of new MPXV mutations warrants the thorough re-investigation of previously recommended or validated primers designed to target MPXV genomes. In this study, we explored 18 PCR primer sets and examined their binding specificity against 5210 MPXV genomes, representing all the established MPXV lineages. Our results indicated that only five primer sets resulted in almost all perfect matches against the targeted MPXV lineages, and the remaining primer sets all contained 1-2 mismatches against almost all the MPXV lineages. We further investigated the mismatched primer-genome pairs and discovered that some of the primers overlapped with poorly sequenced and assembled regions of the MPXV genomes, which are consistent across multiple lineages. However, we identified 173 99% genome-wide conserved regions across all 5210 MPXV genomes, representing 30 lineages/clades with at least 80% lineage-specific consensus for future primer development and primer binding evaluation. This exercise is crucial to ensure that the current detection schemes are robust and serve as a framework for primer evaluation in clinical testing development for other infectious diseases.

Aldosterone regulates vascular gene transcription via oxidative stress-dependent and -independent pathways.

OBJECTIVE: Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease. METHODS AND RESULTS: Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. Quantitative reverse transcription-polymerase chain reaction was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared with the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis. CONCLUSIONS: This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress-dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease, and these pathways may provide novel drug targets to prevent atherosclerosis in humans.

An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis.

The sterol regulatory element binding protein (SREBP) family of transcription activators are critical regulators of cholesterol and fatty acid homeostasis. We previously demonstrated that human SREBPs bind the CREB-binding protein (CBP)/p300 acetyltransferase KIX domain and recruit activator-recruited co-factor (ARC)/Mediator co-activator complexes through unknown mechanisms. Here we show that SREBPs use the evolutionarily conserved ARC105 (also called MED15) subunit to activate target genes. Structural analysis of the SREBP-binding domain in ARC105 by NMR revealed a three-helix bundle with marked similarity to the CBP/p300 KIX domain. In contrast to SREBPs, the CREB and c-Myb activators do not bind the ARC105 KIX domain, although they interact with the CBP KIX domain, revealing a surprising specificity among structurally related activator-binding domains. The Caenorhabditis elegans SREBP homologue SBP-1 promotes fatty acid homeostasis by regulating the expression of lipogenic enzymes. We found that, like SBP-1, the C. elegans ARC105 homologue MDT-15 is required for fatty acid homeostasis, and show that both SBP-1 and MDT-15 control transcription of genes governing desaturation of stearic acid to oleic acid. Notably, dietary addition of oleic acid significantly rescued various defects of nematodes targeted with RNA interference against sbp-1 and mdt-15, including impaired intestinal fat storage, infertility, decreased size and slow locomotion, suggesting that regulation of oleic acid levels represents a physiologically critical function of SBP-1 and MDT-15. Taken together, our findings demonstrate that ARC105 is a key effector of SREBP-dependent gene regulation and control of lipid homeostasis in metazoans.

Global gene expression patterns in the post-pneumonectomy lung of adult mice.

BACKGROUND: Adult mice have a remarkable capacity to regenerate functional alveoli following either lung resection or injury that exceeds the regenerative capacity observed in larger adult mammals. The molecular basis for this unique capability in mice is largely unknown. We examined the transcriptomic responses to single lung pneumonectomy in adult mice in order to elucidate prospective molecular signaling mechanisms used in this species during lung regeneration. METHODS: Unilateral left pneumonectomy or sham thoracotomy was performed under general anesthesia (n = 8 mice per group for each of the four time points). Total RNA was isolated from the remaining lung tissue at four time points post-surgery (6 hours, 1 day, 3 days, 7 days) and analyzed using microarray technology. RESULTS: The observed transcriptomic patterns revealed mesenchymal cell signaling, including up-regulation of genes previously associated with activated fibroblasts (Tnfrsf12a, Tnc, Eln, Col3A1), as well as modulation of Igf1-mediated signaling. The data set also revealed early down-regulation of pro-inflammatory cytokine transcripts and up-regulation of genes involved in T cell development/function, but few similarities to transcriptomic patterns observed during embryonic or post-natal lung development. Immunohistochemical analysis suggests that early fibroblast but not myofibroblast proliferation is important during lung regeneration and may explain the preponderance of mesenchymal-associated genes that are over-expressed in this model. This again appears to differ from embryonic alveologenesis. CONCLUSION: These data suggest that modulation of mesenchymal cell transcriptome patterns and proliferation of S100A4 positive mesenchymal cells, as well as modulation of pro-inflammatory transcriptome patterns, are important during post-pneumonectomy lung regeneration in adult mice.

Association between statin-associated myopathy and skeletal muscle damage.

BACKGROUND: Many patients taking statins often complain of muscle pain and weakness. The extent to which muscle pain reflects muscle injury is unknown. METHODS: We obtained biopsy samples from the vastus lateralis muscle of 83 patients. Of the 44 patients with clinically diagnosed statin-associated myopathy, 29 were currently taking a statin, and 15 had discontinued statin therapy before the biopsy (minimal duration of discontinuation 3 weeks). We also included 19 patients who were taking statins and had no myopathy, and 20 patients who had never taken statins and had no myopathy. We classified the muscles as injured if 2% or more of the muscle fibres in a biopsy sample showed damage. Using reverse transcriptase polymerase chain reaction, we evaluated the expression levels of candidate genes potentially related to myocyte injury. RESULTS: Muscle injury was observed in 25 (of 44) patients with myopathy and in 1 patient without myopathy. Only 1 patient with structural injury had a circulating level of creatine phosphokinase that was elevated more than 1950 U/L (10x the upper limit of normal). Expression of ryanodine receptor 3 was significantly upregulated in patients with biopsy evidence of structural damage (1.7, standard error of the mean 0.3). INTERPRETATION: Persistent myopathy in patients taking statins reflects structural muscle damage. A lack of elevated levels of circulating creatine phosphokinase does not rule out structural muscle injury. Upregulation of the expression of ryanodine receptor 3 is suggestive of an intracellular calcium leak.

Rapid recruitment of temporally distinct vascular gene sets by estrogen.

Cardiovascular disease is the leading cause of mortality for both men and women in developed countries. The sex steroid hormone estrogen is required for normal vascular physiology. Estrogen functions by binding to intracellular estrogen receptors (ER), ERalpha and ERbeta, ligand-activated transcription factors that are expressed in both vascular endothelial and smooth muscle cells. We recently demonstrated that long-term (8 d) estrogen treatment in vivo in mice recruits distinct vascular gene sets mediated by ERalpha and ERbeta and that the promoters from these gene sets are enriched for binding sites of specific transcription factors, leading to the hypothesis that estrogen initiates a cascade of early transcriptional events that modulate gene expression in the vasculature. Here we test this hypothesis using gene expression profiling to examine initial transcriptional events (2-8 h) mediated by estrogen in blood vessels. Our data reveal that 1) estrogen regulates temporally distinct cascades of vascular gene expression, 2) initially, estrogen-mediated vascular gene repression predominates, 3) the earliest estrogen-recruited gene program is enriched in vascular transcription factors that can interact with binding sites present in estrogen-regulated vascular genes recruited subsequently, and 4) estrogen-regulated genes recruited next have specific functions, including lipid metabolism and cellular growth and proliferation that are potentially important for estrogen's known vascular functions. In summary, estrogen directly and rapidly recruits specific transcriptional factors that then propagate distinct cascades of gene expression. These data define the temporal recruitment of specific vascular genes by estrogen and enable further analysis of the mechanisms by which estrogen directly regulates vascular function.

Depletion of central BDNF in mice impedes terminal differentiation of new granule neurons in the adult hippocampus.

Granule neurons generated in the adult mammalian hippocampus synaptically integrate to facilitate cognitive function and antidepressant efficacy. Here, we investigated the role of BDNF in facilitating their maturation in vivo. We found that depletion of central BDNF in mice elicited an increase in hippocampal cell proliferation without affecting cell survival or fate specification. However, new mutant neurons failed to fully mature as indicated by their lack of calbindin, reduced dendritic differentiation and an accumulation of calretinin(+) immature neurons in the BDNF mutant dentate gyrus. Furthermore, the facilitating effects of GABA(A) receptor stimulation on neurogenesis were absent in the mutants, suggesting that defects might be due to alterations in GABA signaling. Transcriptional analysis of the mutant hippocampal neurogenic region revealed increases in markers for immature neurons and decreases in neuronal differentiation facilitators. These findings demonstrate that BDNF is required for the terminal differentiation of new neurons in the adult hippocampus.

Computational modeling and experimental evaluation of a novel prodrug for targeting the extracellular space of prostate tumors.

We are developing a noninvasive approach for targeting imaging and therapeutic radionuclides to prostate cancer. Our method, Enzyme-Mediated Cancer Imaging and Therapy (EMCIT), aims to use enzyme-dependent, site-specific, in vivo precipitation of a radioactive molecule within the extracellular space of solid tumors. Advanced methods for data mining of the literature, protein databases, and knowledge bases (IT. Omics LSGraph and Ingenuity Systems) identified prostatic acid phosphatase (PAP) as an enzyme overexpressed in prostate cancer and secreted in the extracellular space. Using AutoDock 3.0 software, the prodrug ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-P)) was docked in silico into the X-ray structure of PAP. The data indicate that IQ(2-P) docked into the PAP active site with a calculated inhibition constant (K(i)) more favorable than that of the PAP inhibitor alpha-benzylaminobenzylphosphonic acid. When (125)IQ(2-P), the radioiodinated form of the water-soluble prodrug, was incubated with PAP, rapid hydrolysis of the compound was observed as exemplified by formation of the water-insoluble 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone ((125)IQ(2-OH)). Similarly, the incubation of IQ(2-P) with human LNCaP, PC-3, and 22Rv1 prostate tumor cells resulted in the formation of large fluorescent IQ(2-OH) crystals. No hydrolysis was seen in the presence of normal human cells. Autoradiography of tumor cells incubated with (125)IQ(2-P) showed accumulation of radioactive grains ((125)IQ(2-OH)) around the cells. We anticipate that the EMCIT approach will enable the active in vivo entrapment of radioimaging and radiotherapeutic compounds within the extracellular spaces of primary prostate tumors and their metastases.

In silico design, synthesis, and biological evaluation of radioiodinated quinazolinone derivatives for alkaline phosphatase-mediated cancer diagnosis and therapy.

As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap water-insoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2'-phosphoryloxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ(2-P)), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2'-hydroxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ(2-OH)). Biodistribution data suggest the existence of two isoforms of the prodrug (IQ(2-P(I)) and IQ(2-P)), and this has been confirmed by their synthesis and characterization. Structural differences of the two isoforms have been examined using in silico molecular modeling techniques and docking methods to describe the interaction/binding between the isoforms and human placental alkaline phosphatase (PLAP), a tumor cell, membrane-associated, hydrolytic enzyme whose structure is known by X-ray crystallographic determination. Docking data show that IQ(2-P), but not IQ(2-P(I)), fits the active binding site of PLAP favorably and interacts with the catalytic amino acid Ser(92), which plays an important role in the hydrolytic process. The binding free energies (DeltaG(binding)) of the isoforms to PLAP predict that IQ(2-P) will be the better substrate for PLAP. The in vitro incubation of the isoforms with PLAP leads to the rapid hydrolysis of IQ(2-P) only and confirms the in silico expectations. Fluorescence microscopy shows that in vitro incubation of IQ(2-P) with mouse and human tumor cells causes the extracellular, alkaline phosphatase-mediated hydrolysis of the molecule and precipitation of fluorescent crystals of IQ(2-OH). No hydrolysis is seen in the presence of normal mouse and human cells. Furthermore, the intratumoral injection of 125IQ(2-P) into alkaline phosphatase-expressing solid human tumors grown s.c. in nude rats results in efficient hydrolysis of the compound and retention of approximately 70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (approximately 1%) or retention of radioactivity at the injected site. These studies support the enzyme-mediated cancer imaging and therapy technology and show the potential of such quinazolinone derivatives in the in vivo radiodetection (123I/124I) and therapy (131I) of solid tumors.

Unliganded estrogen receptor alpha regulates vascular cell function and gene expression.

The unliganded form of the estrogen receptor is generally thought to be inactive. Our prior studies, however, suggested that unliganded estrogen receptor alpha (ERα) exacerbates adverse vascular injury responses in mice. Here, we show that the presence of unliganded ERα decreases vascular endothelial cell (EC) migration and proliferation, increases smooth muscle cell (SMC) proliferation, and increases inflammatory responses in cultured ECs and SMCs. Unliganded ERα also regulates many genes in vascular ECs and mouse aorta. Activation of ERα by E2 reverses the cell physiological effects of unliganded ERα, and promotes gene regulatory effects that are predicted to counter the effects of unliganded ERα. These results reveal that the unliganded form of ERα is not inert, but significantly impacts gene expression and physiology of vascular cells. Furthermore, they indicate that the cardiovascular protective effects of estrogen may be connected to its ability to counteract these effects of unliganded ERα.

A combined approach to data mining of textual and structured data to identify cancer-related targets.

BACKGROUND: We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez), and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis) was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Proteins were identified with respect to six types of cancer occurring in the prostate, breast, lung, colon, ovary, and pancreas. RESULTS: The data mining method identified previously undetected targets. Our combined strategy applied to each cancer type identified a minimum of 375 proteins expressed within the extracellular space and/or attached to the plasma membrane. The method led to the recognition of human cancer-related hydrolases (on average, approximately 35 per cancer type), among which were prostatic acid phosphatase, prostate-specific antigen, and sulfatase 1. CONCLUSION: The combined data mining of several databases overcame many of the limitations of querying a single database and enabled the facile identification of gene products. In the case of cancer-related targets, it produced a list of putative extracellular, hydrolytic enzymes that merit additional study as candidates for cancer radioimaging and radiotherapy. The proposed data mining strategy is of a general nature and can be applied to other biological databases for understanding biological functions and diseases.

MicroRNA-Offset RNA Alters Gene Expression and Cell Proliferation.

MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a "seed region" of moR-21 as well as a "seed match region" in the target gene 3'UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active.

TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior.

Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia-different classes of glial cells-have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior.