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Here we present and develop the hypothesis that the derepression of endogenous retrotransposable elements (RTEs) - 'genomic parasites' - is an important and hitherto under-unexplored molecular aging process that can potentially occur in most tissues. We further envision that the activation and continued presence of retrotransposition contribute to age-associated tissue degeneration and pathology. Chromatin is a complex and dynamic structure that needs to be maintained in a functional state throughout our lifetime. Studies of diverse species have revealed that chromatin undergoes extensive rearrangements during aging. Cellular senescence, an important component of mammalian aging, has recently been associated with decreased heterochromatinization of normally silenced regions of the genome. These changes lead to the expression of RTEs, culminating in their transposition. RTEs are common in all kingdoms of life, and comprise close to 50% of mammalian genomes. They are tightly controlled, as their activity is highly destabilizing and mutagenic to their resident genomes. [ABSTRACT FROM AUTHOR]

Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity. [ABSTRACT FROM AUTHOR]

Chromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging. [ABSTRACT FROM AUTHOR]

Abstract: Vertebrate embryos generate striking Ca2+ patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca2+ during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca2+ along the dorsal–ventral axis, with higher Ca2+ concentrations in the ventral margin and lower Ca2+ concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca2+ pump with 0.5μM thapsigargin elevates cytosolic Ca2+ in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left–right asymmetry. Brain defects include a left–right reversal of pitx2 expression in the dorsal diencephalon and a left–right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca2+ pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca2+ pump during gastrulation inhibits expression of no tail (ntl) and left–right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer’s vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca2+ plays a role in Kupffer’s vesicle function, influencing ciliary motility and translating the vesicle’s counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca2+ plays an additional role in the formation of Kupffer’s vesicle. [Copyright &y& Elsevier]

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions. [ABSTRACT FROM AUTHOR]

Abstract: Using the surf clam embryo, we investigated the effects of the combination of bromoform, chloroform, and tetrachloroethylene, three pollutants found in high concentrations in the municipal water supply in Brick, New Jersey. Exposure produced an increase in an isoform of the regulatory subunit (RII) of cAMP-dependent protein kinase, demonstrated by confocal microscopy and western blotting. Embryos showed an increase in RII where the primordial gill and ciliated velar epithelium are innervated. This increase correlated with increased ciliary activity, indicating a corresponding rise in the catalytic subunit. Treatment resulted in decreased threonine phosphorylation of actin. There was no effect on neurotransmitters or receptors of the serotonergic–dopaminergic nervous system. These effects occurred only with the ternary mixture. No significant effect was seen with individual or paired components. This is the first report showing that bromoform, chloroform, and tetrachloroethylene act synergistically to alter a key regulator of neuronal development. [Copyright &y& Elsevier]

The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike dam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R² = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs. Key words: neurotoxicology, p63, p73, PCBs, surf clam, Spisula solidissima. Environ Health Perspect 110:377–385 (2002). [Online 7 March 2002] [ABSTRACT FROM AUTHOR]

Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders. [Display omitted] • Evaluation of zebrafish larval behavior using a high throughput imaging system. • Exposure of larvae to 876 FDA-approved drugs yielded complex behavioral profiles. • Identified 64 cyclosporine-like compounds with potential neuroprotective effects. [ABSTRACT FROM AUTHOR]

Investigates the antimicrobial actions of Limulus C-reactive proteins (CRP) and compares them to those of Limulus anti-lipopolyssacharide factor (LALF). LALF's and CRP's effect on Vibrio alginolyticus and Escherichia coli; Observation on CRP's bacteriostatic and LALF's bacteriolytic effect.

Reports on the neurotoxicity of polychlorinated biphenyls in the developing Spisula solidissima embryo. Decrease in serotonin production upon exposure; Delay in onset and cessation of expression of a molluscan-specific neural antigen.

RNA localization in the Xenopus oocyte is responsible for the establishment of polarity during oogenesis as well as the specification of germ layers during embryogenesis. However, the inability to monitor mRNA localization in live vertebrate oocytes has posed a major barrier to understanding the mechanisms driving directional transport. Here we describe a method for imaging MS2 tagged RNA in live Xenopus oocytes to study the dynamics of RNA localization. We also focus on methods for implementing and analyzing FRAP data. This protocol is optimized for imaging of the RNAs in stage II oocytes but it can be adapted to study dynamics of other molecules during oogenesis. Using this approach, mobility can be measured in different regions of the oocyte, enabling the direct observation of molecular dynamics throughout the oocyte. [ABSTRACT FROM AUTHOR]

Behavioral testing is an essential tool for evaluating cognitive function and dysfunction in preclinical research models. This is of special importance in the study of neurological disorders such as Alzheimer's disease. However, the reproducibility of classic behavioral assays is frequently compromised by interstudy variation, leading to ambiguous conclusions about the behavioral markers characterizing the disease. Here, we identify age- and genotype-driven differences between 3xTg-AD and non-transgenic control mice using a low-cost, highly customizable behavioral assay that requires little human intervention. Through behavioral phenotyping combining both supervised and unsupervised behavioral classification methods, we are able to validate the preventative effects of the immunosuppressant cyclosporine A in a rodent model of Alzheimer's disease, as well as the partially ameliorating effects of candidate drugs nebivolol and cabozantinib. [Display omitted] • Behavioral assays are an important tool to assess cognitive function in pre-clinical neurological disease models. • We combined supervised and unsupervised behavioral classification methods to quantify deficits in female 3xTg-AD mice. • Chronic treatment with cyclosporine, nebivolol, or cabozantinib mitigate behavioral and gait deficits in young and old mice. [ABSTRACT FROM AUTHOR]

Summary: RNA localization is a widely conserved mechanism for generating cellular asymmetry. In Xenopus oocytes, microtubule-dependent transport of RNAs to the vegetal cortex underlies germ layer patterning. Although kinesin motors have been implicated in this process, the apparent polarity of the microtubule cytoskeleton has pointed instead to roles for minus-end-directed motors. To resolve this issue, we have analyzed participation of kinesin motors in vegetal RNA transport and identified a direct role for Xenopus kinesin-1. Moreover, in vivo interference and biochemical experiments reveal a key function for multiple motors, specifically kinesin-1 and kinesin-2, and suggest that these motors may interact during transport. Critically, we have discovered a subpopulation of microtubules with plus ends at the vegetal cortex, supporting roles for these kinesin motors in vegetal RNA transport. These results provide a new mechanistic basis for understanding directed RNA transport within the cytoplasm. [Copyright &y& Elsevier]

Repurposing FDA-approved drugs is an efficient and cost-effective approach in the development of therapeutics for a broad range of diseases. However, prediction of function can be challenging, especially in the brain. We screened a small-molecule library with FDA-approved drugs for effects on behavior. The studies were carried out using zebrafish larvae, imaged in a 384-well format. We found that various drugs affect activity, habituation, startle responses, excitability, and optomotor responses. The changes in behavior were organized in behavioral profiles, which were examined by hierarchical cluster analysis. One of the identified clusters includes the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506), which are immunosuppressants and potential therapeutics in the prevention of Alzheimer's disease. The calcineurin inhibitors form a functional cluster with seemingly unrelated drugs, including bromocriptine, tetrabenazine, rosiglitazone, nebivolol, sorafenib, cabozantinib, tamoxifen, meclizine, and salmeterol. We propose that drugs with 'CsA-type' behavioral profiles are promising candidates for the prevention and treatment of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age-associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer-lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well-characterized processes. In particular, understanding the role of sex-determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs. [ABSTRACT FROM AUTHOR]

Replicative cellular senescence is an important tumor suppression mechanism and also contributes to aging. Progression of both cancer and aging include significant epigenetic components, but the chromatin changes that take place during cellular senescence are not known. We used formaldehyde assisted isolation of regulatory elements ( FAIRE) to map genome-wide chromatin conformations. In contrast to growing cells, whose genomes are rich with features of both open and closed chromatin, FAIRE profiles of senescent cells are significantly smoothened. This is due to FAIRE signal loss in promoters and enhancers of active genes, and FAIRE signal gain in heterochromatic gene-poor regions. Chromatin of major retrotransposon classes, Alu, SVA and L1, becomes relatively more open in senescent cells, affecting most strongly the evolutionarily recent elements, and leads to an increase in their transcription and ultimately transposition. Constitutive heterochromatin in centromeric and peri-centromeric regions also becomes relatively more open, and the transcription of satellite sequences increases. The peripheral heterochromatic compartment ( PHC) becomes less prominent, and centromere structure becomes notably enlarged. These epigenetic changes progress slowly after the onset of senescence, with some, such as mobilization of retrotransposable elements becoming prominent only at late times. Many of these changes have also been noted in cancer cells. [ABSTRACT FROM AUTHOR]

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ∼0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons. [ABSTRACT FROM AUTHOR]

[Display omitted] • Zebrafish larval behavior can be imaged in vivo at an in vitro scale. • Modulators of calcineurin signaling affect behavior. • Our studies revealed beneficial and adverse effects of small-molecule therapeutics. • The findings have clinical relevance in Down syndrome and Alzheimer's disease. Small-molecule modulators of calcineurin signaling have been proposed as potential therapeutics in Down syndrome and Alzheimer's disease. Models predict that in Down syndrome, suppressed calcineurin-NFAT signaling may be mitigated by proINDY, which activates NFAT, the nuclear factor of activated T-cells. Conversely, elevated calcineurin signaling in Alzheimer's disease may be suppressed with the calcineurin inhibitors cyclosporine and tacrolimus. Such small-molecule treatments may have both beneficial and adverse effects. The current study examines the effects of proINDY, cyclosporine and tacrolimus on behavior, using zebrafish larvae as a model system. To suppress calcineurin signaling, larvae were treated with cyclosporine and tacrolimus. We found that these calcineurin inhibitors induced hyperactivity, suppressed visually-guided behaviors, acoustic hyperexcitability and reduced habituation to acoustic stimuli. To activate calcineurin-NFAT signaling, larvae were treated with proINDY. ProINDY treatment reduced activity and stimulated visually-guided behaviors, opposite to the behavioral changes induced by calcineurin inhibitors. The opposing effects suggest that activity and visually-guided behaviors are regulated by the calcineurin-NFAT signaling pathway. A central role of calcineurin-NFAT signaling is further supported by co-treatments of calcineurin inhibitors and proINDY, which had therapeutic effects on activity and visually-guided behaviors. However, these co-treatments adversely increased excitability, suggesting that some behaviors are regulated by other calcineurin signaling pathways. Overall, the developed methodologies provide an efficient high-throughput platform for the evaluation of modulators of calcineurin signaling that restore neural function, while avoiding adverse side effects, in a complex neural system. [ABSTRACT FROM AUTHOR]

We analyzed the small RNA transcriptome from 5‐month‐old, 24‐month‐old, and 36‐month‐old mouse liver and found 56 miRNAs that changed their expression profile with age. Among these is a cluster of 18 miRNAs that are upregulated between 50‐ and 1,000‐fold at 24 and 36 months of age. This cluster is located in a 60‐kb region of the X‐chromosome that is devoid of other coding sequences and is part of a lamin‐associated domain. Potential targets of the miRNAs in the cluster suggest they may regulate several pathways altered in aging, including the PI3K‐Akt pathway. Total transcriptome analyses indicate that expression of several potential genes in the PI3K‐Akt pathway that may be targeted by the mir‐465 family (mmu‐mir‐465a, mmu‐mir‐465b, and mmu‐mir‐465c) is downregulated with age. Transfection of the liver cell line AML12 with mir‐465 family members leads to a reduction of three of these potential targets at the mRNA level: a 40% reduction of the growth hormone receptor (GHR), and a 25% reduction in Kitl and PPP2R3C. Further investigation of the GHR 3′UTR revealed that the mir‐465 family directly targets the GHR mRNA. Cells transfected with mir‐465 showed a reduction of JAK2 and STAT5 phosphorylation upon growth hormone (GH) stimulation, resulting in a reduction in insulin‐like growth factor 1 (IGF‐1) and IGF‐1‐binding protein 3 expression. With age, GH signaling falls and there is a reduction in circulating IGF‐1. Our data suggest that an increase in expression of the mir‐465 family with age may contribute to the reduction in the GH signaling. [ABSTRACT FROM AUTHOR]

An abstract of an article on how notch signalling with the help of ciliary length control regulates left-right asymmetry is presented.