Mother-daughter centriole disengagement, the necessary first step in centriole duplication, involves Plk1 activity in early mitosis and separase activity after APC/C activity mediates securin degradation. Plk1 activity is thought to be essential and sufficient for centriole disengagement with separase activity playing a supporting but non-essential role. In separase null cells, however, centriole disengagement is substantially delayed. The ability of APC/C activity alone to mediate centriole disengagement has not been directly tested. We investigate the interrelationship between Plk1 and APC/C activities in disengaging centrioles in S or G2 HeLa and RPE1 cells, cell types that do not reduplicate centrioles when arrested in S phase. Knockdown of the interphase APC/C inhibitor Emi1 leads to centriole disengagement and reduplication of the mother centrioles, though this is slow. Strong inhibition of Plk1 activity, if any, during S does not block centriole disengagement and mother centriole reduplication in Emi1 depleted cells. Centriole disengagement depends on APC/C-Cdh1 activity, not APC/C-Cdc20 activity. Also, Plk1 and APC/C-Cdh1 activities can independently promote centriole disengagement in G2 arrested cells. Thus, Plk1 and APC/C-Cdh1 activities are independent but slow pathways for centriole disengagement. By having two slow mechanisms for disengagement working together, the cell ensures that centrioles will not prematurely separate in late G2 or early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion only late in mitosis.
Vomeronasal sensory neurons (VSNs) extend axons to the accessory olfactory bulb (AOB) where they form synaptic connections that relay pheromone signals to the brain. The projections of apical and basal VSNs segregate in the AOB into anterior (aAOB) and posterior (pAOB) compartments. Although some aspects of this organization exhibit fundamental similarities with the main olfactory system, the mechanisms that regulate mammalian vomeronasal targeting are not as well understood. In the olfactory epithelium (OE), the glycosyltransferase beta3GnT2 maintains expression of axon guidance cues required for proper glomerular positioning and neuronal survival. We show here that beta3GnT2 also regulates guidance and adhesion molecule expression in the vomeronasal system in ways that are partially distinct from the OE. In wildtype mice, ephrinA5(+) axons project to stereotypic subdomains in both the aAOB and pAOB compartments. This pattern is dramatically altered in beta3GnT2(-/-) mice, where ephrinA5 is upregulated exclusively on aAOB axons. Despite this, apical and basal VSN projections remain strictly segregated in the null AOB, although some V2r1b axons that normally project to the pAOB inappropriately innervate the anterior compartment. These fibers appear to arise from ectopic expression of V2r1b receptors in a subset of apical VSNs. The homotypic adhesion molecules Kirrel2 and OCAM that facilitate axon segregation and glomerular compartmentalization in the main olfactory bulb are ablated in the beta3GnT2(-/-) aAOB. This loss is accompanied by a two-fold increase in the total number of V2r1b glomeruli and a failure to form morphologically distinct glomeruli in the anterior compartment. These results identify a novel function for beta3GnT2 glycosylation in maintaining expression of layer-specific vomeronasal receptors, as well as adhesion molecules required for proper AOB glomerular formation.
The bacterial lacZ gene is widely used as a reporter in a myriad of mouse transgenic experiments. beta-Galactosidase, encoded by lacZ, is usually detected using X-gal in combination with ferric and ferrous ions. This assay produces a blue indole precipitate that is easy to detect visually. Here, we show that Salmon-gal in combination with tetrazolium salts provides a more sensitive and faster staining reaction than the traditional beta-galactosidase assay in mouse embryos. Using a combination of Salmon-gal and tetranitroblue tetrazolium, we were able to visualize the activity of beta-galactosidase in embryos at stages when the customary X-gal reaction failed to detect staining. Our studies provide an enhanced alternative for beta-galactosidase detection in expression and cell fate studies that use lacZ-based transgenic mouse lines.
UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5' end of a nuclear mRNA, but are also reported to associate with the Exon Junction Complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly we used Fluorescence Recovery after Photobleaching (FRAP) to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the PI3 kinase / AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 Kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intron-less Histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the Endoplasmic Reticulum or to Mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and can promote the nuclear retention of some mRNAs.
Integrin-based adhesions promote cell survival as well as cell motility and invasion. We show here that the adhesion regulatory protein supervillin increases cell survival by decreasing levels of the tumor suppressor protein p53 and downstream target genes. RNAi-mediated knockdown of a new splice form of supervillin (isoform 4) or both isoforms 1 and 4 increases the amount of p53 and cell death, whereas p53 levels decrease after overexpression of either supervillin isoform. Cellular responses to DNA damage induced by etoposide or doxorubicin include down-regulation of endogenous supervillin coincident with increases in p53. In DNA-damaged supervillin knockdown cells, p53 knockdown or inhibition partially rescues the loss of cell metabolic activity, a measure of cell proliferation. Knockdown of the p53 deubiquitinating enzyme USP7/HAUSP also reverses the supervillin phenotype, blocking the increase in p53 levels seen after supervillin knockdown and accentuating the decrease in p53 levels triggered by supervillin overexpression. Conversely, supervillin overexpression decreases the association of USP7 and p53 and attenuates USP7-mediated p53 deubiquitination. USP7 binds directly to the supervillin N terminus and can deubiquitinate and stabilize supervillin. Supervillin also is stabilized by derivatization with the ubiquitin-like protein SUMO1. These results show that supervillin regulates cell survival through control of p53 levels and suggest that supervillin and its interaction partners at sites of cell-substrate adhesion constitute a locus for cross-talk between survival signaling and cell motility pathways.
Genetic modification of the association between peripubertal dioxin exposure and pubertal onset in a cohort of Russian boys
BACKGROUND: Exposure to dioxins has been associated with delayed pubertal onset in both epidemiologic and animal studies. Whether genetic polymorphisms may modify this association is currently unknown. Identifying such genes could provide insight into mechanistic pathways. This is one of the first studies to assess genetic susceptibility to dioxins.
OBJECTIVES: We evaluated whether common polymorphisms in genes affecting either molecular responses to dioxin exposure or pubertal onset influence the association between peripubertal serum dioxin concentration and male pubertal onset.
METHODS: In this prospective cohort of Russian adolescent boys (n = 392), we assessed gene-environment interactions for 337 tagging single-nucleotide polymorphisms (SNPs) from 46 candidate genes and two intergenic regions. Dioxins were measured in the boys' serum at age 8-9 years. Pubertal onset was based on testicular volume and on genitalia staging. Statistical approaches for controlling for multiple testing were used, both with and without prescreening for marginal genetic associations.
RESULTS: After accounting for multiple testing, two tag SNPs in the glucocorticoid receptor (GR/NR3C1) gene and one in the estrogen receptor-alpha (ESR1) gene were significant (q < 0.2) modifiers of the association between peripubertal serum dioxin concentration and male pubertal onset defined by genitalia staging, although not by testicular volume. The results were sensitive to whether multiple comparison adjustment was applied to all gene-environment tests or only to those with marginal genetic associations.
CONCLUSIONS: Common genetic polymorphisms in the glucocorticoid receptor and estrogen receptor-alpha genes may modify the association between peripubertal serum dioxin concentration and pubertal onset. Further studies are warranted to confirm these findings.
A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures
Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.
Nuclear Shape Changes Are Induced by Knockdown of the SWI/SNF ATPase BRG1 and Are Independent of Cytoskeletal Connections
Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.
A stereological study of the numbers of neurons and glia in the primary visual cortex across the lifespan of male and female rhesus monkeys
Mild age-related declines in visual function occur in humans and monkeys, independent of ocular pathology, suggesting involvement of central visual pathways (Spear  Vision Res 33:2589-2609). Although many factors might account for this decline, a loss of neurons in primary visual cortex (V1) could be a contributing factor. Previous studies of neuron numbers in V1 reported stability across age, but were limited in the ages and genders studied and sampled only limited parts of V1 or limited cell types, allowing for the possibility of a subtle loss of neurons. We pursued this question in 26 behaviorally tested adult male and female rhesus monkeys ranging from 7.4 to 31.0 years of age by using design-based stereology to estimate numbers of NeuN-labeled neurons and thionin-stained glia within three laminar zones, supragranular (layers II-IVB), granular (IVC), and infragranular (V-VI), across the entirety of V1. There were no significant differences between males and females on any measures, except for total brain weight (P = 0.0038). There was an average of 416,000,000 neurons in V1, but no effect of age on this total or numbers within any laminar zone. Similarly, there was an average of 184,000,000 glia in V1 (44% of the number of neurons), but no effect of age on this total. However, there was a significant age-related increase in numbers of glia in the infragranular zone, perhaps reflecting a glial response to pathology in myelinated projection fibers. This study provides further evidence that in normal aging neurons are not lost and hence cannot account for age-related dysfunction.
We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.
Pulmonary delivery of d-methionine is associated with an increase in ALCAR and glutathione in cochlear fluids
In animals, hearing loss resulting from cochlear mechanosensory cell damage can be mitigated by antioxidants such as d-methionine (d-met) and acetyl-l-carnitine (ALCAR). The systemic routes of administration of these compounds, that must of necessity transit trough the cochlear fluids, may affect the antioxidant levels in the cochlea and the resulting oto-protective effect. In this study, we analyzed the pharmacokinetics of [C]d-met in the cochlea and four other tissues after intratracheal (IT), intranasal (IN), and oral by gavage (OG) administration and compared it to intravenous administration (IV). We then analyzed the effect of these four routes on the antioxidant content of the cochlear fluids after d-met or ALCAR administration, by liquid chromatography/mass spectrometry. Our results showed that the concentration of methionine and ALCAR in cochlear fluids significantly increased after their respective systemic administration. Interestingly, d-met administration also contributed to an increase of ALCAR. Our results also showed that the delivery routes differently affected the bioavailability of administered [C]d-met as well as the concentrations of methionine, ALCAR and the ratio of oxidized to reduced glutathione. Overall, pulmonary delivery via IT administration achieved high concentrations of methionine, ALCAR, and oxidative-related metabolites in cochlear fluids, in some cases surpassing IV administration, while IN route appeared to be the least efficacious. To our knowledge, this is the first report of the direct measurements of antioxidant levels in cochlear fluids after their systemic administration. This report also demonstrates the validity of the pulmonary administration of antioxidants and highlights the different contributions of d-met and ALCAR allowing to further investigate their impact on oxidative stress in the cochlear microenvironment.
Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio)
Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.
Dietary composition has major effects on physiology. Here, we show that developmental rate, reproduction, and lifespan are altered in C. elegans fed Comamonas DA1877 relative to those fed a standard E. coli OP50 diet. We identify a set of genes that change in expression in response to this diet and use the promoter of one of these (acdh-1) as a dietary sensor. Remarkably, the effects on transcription and development occur even when Comamonas DA1877 is diluted with another diet, suggesting that Comamonas DA1877 generates a signal that is sensed by the nematode. Surprisingly, the developmental effect is independent from TOR and insulin signaling. Rather, Comamonas DA1877 affects cyclic gene expression during molting, likely through the nuclear hormone receptor NHR-23. Altogether, our findings indicate that different bacteria elicit various responses via distinct mechanisms, which has implications for diseases such as obesity and the interactions between the human microbiome and intestinal cells.
A compendium of Caenorhabditis elegans RNA binding proteins predicts extensive regulation at multiple levels
Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs.
Gene families expand by gene duplication and resulting paralogs diverge through mutation. Functional diversification can include neo-functionalization as well as sub-functionalization of ancestral functions. In addition, redundancy in which multiple genes fulfill overlapping functions is often maintained. Here, we use the family of 40 Caenorhabditis elegans insulins to gain insight into the balance between specificity and redundancy. The insulin/insulin-like growth factor (IIS) pathway comprises a single receptor, DAF-2. To date, no single insulin-like peptide recapitulates all DAF-2-associated phenotypes, likely due to redundancy between insulin-like genes. To provide a first-level annotation of potential patterns of redundancy, we comprehensively delineate the spatiotemporal and conditional expression of all 40 insulins in living animals. We observe extensive dynamics in expression that can explain the lack of simple patterns of pair-wise redundancy. We propose a model in which gene families evolve to attain differential alliances in different tissues and in response to a range of environmental stresses.
Expression profiles are tailored according to dietary input. However, the networks that control dietary responses remain largely uncharacterized. Here, we combine forward and reverse genetic screens to delineate a network of 184 genes that affect the C. elegans dietary response to Comamonas DA1877 bacteria. We find that perturbation of a mitochondrial network composed of enzymes involved in amino acid metabolism and the TCA cycle affects the dietary response. In humans, mutations in the corresponding genes cause inborn diseases of amino acid metabolism, most of which are treated by dietary intervention. We identify several transcription factors (TFs) that mediate the changes in gene expression upon metabolic network perturbations. Altogether, our findings unveil a transcriptional response system that is poised to sense dietary cues and metabolic imbalances, illustrating extensive communication between metabolic networks in the mitochondria and gene regulatory networks in the nucleus.
Mammalian genomes encode genetic information in their linear sequence, but appropriate expression of their genes requires chromosomes to fold into complex three-dimensional structures. Transcriptional control involves the establishment of physical connections among genes and regulatory elements, both along and between chromosomes. Recent technological innovations in probing the folding of chromosomes are providing new insights into the spatial organization of genomes and its role in gene regulation. It is emerging that folding of large complex chromosomes involves a hierarchy of structures, from chromatin loops that connect genes and enhancers to larger chromosomal domains and nuclear compartments. The larger these structures are along this hierarchy, the more stable they are within cells, while becoming more stochastic between cells. Here, we review the experimental and theoretical data on this hierarchy of structures and propose a key role for the recently discovered topologically associating domains.
Objectives: (1) to identify common errors in data organization and metadata completeness that would preclude a “reader” from being able to interpret and re-use the data for a new purpose; and (2) to develop a set of best practices derived from these common errors that would guide researchers in creating more usable data products that could be readily shared, interpreted, and used.
Methods: We used directed qualitative content analysis to assess and categorize data and metadata errors identified by peer reviewers of data papers published in the Ecological Society of America’s (ESA) Ecological Archives. Descriptive statistics provided the relative frequency of the errors identified during the peer review process.
Results: There were seven overarching error categories: Collection & Organization, Assure, Description, Preserve, Discover, Integrate, and Analyze/Visualize. These categories represent errors researchers regularly make at each stage of the Data Life Cycle. Collection & Organization and Description errors were some of the most common errors, both of which occurred in over 90% of the papers.
Conclusions: Publishing data for sharing and reuse is error prone, and each stage of the Data Life Cycle presents opportunities for mistakes. The most common errors occurred when the researcher did not provide adequate metadata to enable others to interpret and potentially re-use the data. Fortunately, there are ways to minimize these mistakes through carefully recording all details about study context, data collection, QA/ QC, and analytical procedures from the beginning of a research project and then including this descriptive information in the metadata.
Phone-delivered Mindfulness Training for Patients with Implantable Cardioverter Defibrillators: Results of a Pilot Randomized Controlled Trial
The reduction in adrenergic activity and anxiety associated with meditation may be beneficial for patients with implantable cardioverter defibrillators. Purpose
This study aims to determine the feasibility of a phone-delivered mindfulness intervention in patients with defibrillators and to obtain preliminary indications of efficacy on mindfulness and anxiety. Methods
Clinically stable outpatients were randomized to a mindfulness intervention (eight weekly individual phone sessions) or to a scripted follow-up phone call. We used the Hospital Anxiety and Depression Scale and the Five Facets of Mindfulness to measure anxiety and mindfulness, and multivariate linear regression to estimate the intervention effect on pre-post-intervention changes in these variables. Results
We enrolled 45 patients (23 mindfulness and 22 control; age, 43–83; 30 % women). Retention was 93 %; attendance was 94 %. Mindfulness (beta = 3.31; p = 0.04) and anxiety (beta = −1.15; p = 0.059) improved in the mindfulness group. Conclusions
Mindfulness training can be effectively phone-delivered and may improve mindfulness and anxiety in cardiac defibrillator outpatients.
A therapeutic dose of ketoprofen causes acute gastrointestinal bleeding, erosions, and ulcers in rats
Perioperative treatment of several rats in our facility with ketoprofen (5 mg/kg SC) resulted in blood loss, peritonitis, and death within a day to a little more than a week after surgery that was not related to the gastrointestinal tract. Published reports have established the 5-mg/kg dose as safe and effective for rats. Because ketoprofen is a nonselective nonsteroidal antiinflammatory drug that can damage the gastrointestinal tract, the putative diagnosis for these morbidities and mortalities was gastrointestinal toxicity caused by ketoprofen (5 mg/kg). We conducted a prospective study evaluating the effect of this therapeutic dose of ketoprofen on the rat gastrointestinal tract within 24 h. Ketoprofen (5 mg/kg SC) was administered to one group of rats that then received gas anesthesia for 30 min and to another group without subsequent anesthesia. A third group was injected with saline followed by 30 min of gas anesthesia. Our primary hypothesis was that noteworthy gastrointestinal bleeding and lesions would occur in both groups treated with ketoprofen but not in rats that received saline and anesthesia. Our results showed marked gastrointestinal bleeding, erosions, and small intestinal ulcers in the ketoprofen-treated rats and minimal damages in the saline-treated group. The combination of ketoprofen and anesthesia resulted in worse clinical signs than did ketoprofen alone. We conclude that a single 5-mg/kg dose of ketoprofen causes acute mucosal damage to the rat small intestine.