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In JoVE (1)

Other Publications (13)

Articles by Julie A. Williams in JoVE

 JoVE Immunology and Infection

Quantitative Measurement of the Immune Response and Sleep in Drosophila

1Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine


JoVE 4355

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.

Other articles by Julie A. Williams on PubMed

The Maintenance Diets of C57BL/6J and 129X1/SvJ Mice Influence Their Taste Solution Preferences: Implications for Large-scale Phenotyping Projects

We examined the extent to which maintenance diet influences the taste preferences of mice. C57BL/6J (B6) and 129X1/SvJ (129) mice were fed one of three standard cereal-based diets (Teklad 8604, Zeigler NIH-07, Purina 5001), a cereal-based diet formulated for breeding (Purina 5015), or two purified diets (AIN-76A or AIN-93G). The mice were given 48-h two-bottle choice tests between water and the following seven taste solutions: 2 mmol/L saccharin, 5 mmol/L citric acid, 50 mmol/L citric acid, 30 micro mol/L quinine hydrochloride (QHCl), 300 micro mol/L QHCl, 75 mmol/L NaCl, and 10% ethanol. There were very few differences in taste solution preference scores among mice of the same strain fed the three different versions of standard cereal-based diet. There were also very few differences in taste solution preference scores between mice of the same strain fed the two purified diets. However, the mice fed standard cereal-based diets generally drank more water and total fluid than did mice fed purified diets. There were larger differences between the B6 and 129 strains in saccharin and ethanol preference scores with mice fed standard cereal-based diets than purified diets. Conversely, there were larger differences between the B6 and 129 strains in citric acid and NaCl preference scores with mice fed purified diets than standard cereal-based diets. These results show that maintenance diet composition can have strain-dependent effects on taste solution preference. They illustrate that attention must be paid to the effects of diet on phenotype in screens of mutagenized mice and other genetic studies.

Constructing a Feedback Loop with Circadian Clock Molecules from the Silkmoth, Antheraea Pernyi

Circadian clocks are important regulators of behavior and physiology. The circadian clock of Drosophila depends on an autoinhibitory feedback loop involving dCLOCK, CYCLE (also called dBMAL, for Drosophila brain and muscle ARNT-like protein), dPERIOD, and dTIMELESS. Recent studies suggest that the clock mechanism in other insect species may differ strikingly from that of Drosophila. We cloned Clock, Bmal, and Timeless homologs (apClock, apBmal, and apTimeless) from the silkmoth Antheraea pernyi, from which a Period homolog (apPeriod) has already been cloned. In Schneider 2 (S2) cell culture assays, apCLOCK:apBMAL activates transcription through an E-box enhancer element found in the 5' region of the apPeriod gene. Furthermore, apPERIOD can robustly inhibit apCLOCK: apBMAL-mediated transactivation, and apTIMELESS can augment this inhibition. Thus, a complete feedback loop, resembling that found in Drosophila, can be constructed from silkmoth CLOCK, BMAL, PERIOD, and TIMELESS. Our results suggest that the circadian autoinhibitory feedback loop discovered in Drosophila is likely to be widespread among insects. However, whereas the transactivation domain in Drosophila lies in the C terminus of dCLOCK, in A. pernyi, it lies in the C terminus of apBMAL, which is highly conserved with the C termini of BMALs in other insects (except Drosophila) and in vertebrates. Our analysis sheds light on the molecular function and evolution of clock genes in the animal kingdom.

Transcription Factor ERG and Joint and Articular Cartilage Formation During Mouse Limb and Spine Skeletogenesis

Articular cartilage and synovial joints are critical for skeletal function, but the mechanisms regulating their development are largely unknown. In previous studies we found that the ets transcription factor ERG and its alternatively-spliced variant C-1-1 have roles in joint formation in chick. Here, we extended our studies to mouse. We found that ERG is also expressed in developing mouse limb joints. To test regulation of ERG expression, beads coated with the joint master regulator protein GDF-5 were implanted close to incipient joints in mouse limb explants; this led to rapid and strong ectopic ERG expression. We cloned and characterized several mammalian ERG variants and expressed a human C-1-1 counterpart (hERG3Delta81) throughout the cartilaginous skeleton of transgenic mice, using Col2a1 gene promoter/enhancer sequences. The skeletal phenotype was severe and neonatal lethal, and the transgenic mice were smaller than wild type littermates and their skeletons were largely cartilaginous. Limb long bone anlagen were entirely composed of chondrocytes actively expressing collagen IX and aggrecan as well as articular markers such as tenascin-C. Typical growth plates were absent and there was very low expression of maturation and hypertrophy markers, including Indian hedgehog, collagen X and MMP-13. The results suggest that ERG is part of molecular mechanisms leading chondrocytes into a permanent developmental path and become joint forming cells, and may do so by acting downstream of GDF-5.

Interaction Between Sleep and the Immune Response in Drosophila: a Role for the NFkappaB Relish

The regulation of sleep is poorly understood. While some molecules, including those involved in inflammatory/immune responses, have been implicated in the control of sleep, their role in this process remains unclear. The Drosophila model for sleep provides a powerful system to identify and test the role of sleep-relevant molecules.

Feasibility of a Home Constraint-induced Movement Therapy for Hand Weakness After Stroke

Updates on the Use of Non-invasive Brain Stimulation in Physical and Rehabilitation Medicine

Brain stimulation for the treatment of neuropsychiatric diseases has been used for more than 50 years. Although its development has been slow, current advances in the techniques of brain stimulation have improved its clinical efficacy. The use of non-invasive brain stimulation has significant advantages, such as not involving surgical procedures and having relatively mild adverse effects. In this paper we briefly review the use of 2 non-invasive brain stimulation techniques, repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS), as therapeutic approaches in physical and rehabilitation medicine. We also compare the effects of non-invasive central nervous system stimulation with techniques of non-invasive peripheral electrical stimulation, in order to provide new insights for future developments. Although the outcomes of these initial trials include some conflicting results, the evidence supports that rTMS and tDCS might have a therapeutic value in different neurological conditions. Studies published within the last year have examined new approaches of stimulation, such as longer intensities of stimulation, new electrode sizes for tDCS, novel coils for stimulation of deeper areas, and new frequencies of stimulation for rTMS. These new approaches need to be tested in larger clinical trials in order to determine whether they offer significant clinical effects.

Retinoic Acid Receptors Are Required for Skeletal Growth, Matrix Homeostasis and Growth Plate Function in Postnatal Mouse

The retinoic acid receptors alpha, beta and gamma (RARalpha, RARbeta and RARgamma) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARalpha and RARgamma (or RARbeta and RARgamma) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARalpha and RARbeta, however, are virtually normal, suggesting that RARgamma is essential. In good correlation, we find that RARgamma is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARgamma is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARgamma over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARgamma in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.

Inhibition of Ectopic Bone Formation by a Selective Retinoic Acid Receptor Alpha-agonist: a New Therapy for Heterotopic Ossification?

Heterotopic ossification (HO) consists of formation of ectopic cartilage followed by endochondral bone and is triggered by major surgeries, large wounds, and other conditions. Current therapies, including low-dose irradiation, are not always effective and do not target the skeletogenic process directly. Because chondrogenesis requires a decrease of nuclear retinoic acid receptor alpha (RARalpha) action, we reasoned that pharmacologic activation of this receptor pathway should inhibit HO. Thus, we selected the synthetic retinoid NRX195183, a potent and highly selective RARalpha-agonist, and found that it did inhibit chondrogenesis in mouse limb micromass cultures. We established a mouse HO model consisting of subcutaneous implantation of Matrigel mixed with rhBMP-2. Control mice receiving daily oral doses of vehicle (peanut oil) or retinol (a natural nonactive retinoid precursor) developed large HO-like masses by days 9-12 that displayed abundant cartilage, endochondral bone, vessels, and marrow. In contrast, formation of HO-like masses was markedly reduced in companion mice receiving daily oral doses of alpha-agonist. These ectopic masses contained sharply reduced amounts of cartilage and bone, blood vessels, and TRAP-positive osteoclasts, and expressed markedly lower levels of master chondrogenic genes including Sox9, cartilage genes such as collagen XI and X, and osteogenic genes including Runx2. The data provide proof-of-principle evidence that a pharmacological strategy involving a selective RARalpha-agonist can indeed counteract an ectopic skeletal-formation process effectively and efficiently, and could thus represent a novel preventive treatment for HO.

Wnt/beta-catenin and Retinoic Acid Receptor Signaling Pathways Interact to Regulate Chondrocyte Function and Matrix Turnover

Activation of the Wnt/beta-catenin and retinoid signaling pathways is known to tilt cartilage matrix homeostasis toward catabolism. Here, we investigated possible interactions between these pathways. We found that all-trans-retinoic acid (RA) treatment of mouse epiphyseal chondrocytes in culture did increase Wnt/beta-catenin signaling in the absence or presence of exogenous Wnt3a, as revealed by lymphoid enhancer factor/T-cell factor/beta-catenin reporter activity and beta-catenin nuclear accumulation. This stimulation was accompanied by increased gene expression of Wnt proteins and receptors and was inhibited by co-treatment with Dickkopf-related protein-1, an extracellular inhibitor of Wnt/beta-catenin signaling, suggesting that RA modulates Wnt signaling at Wnt cell surface receptor level. RA also enhanced matrix loss triggered by Wnt/beta-catenin signaling, whereas treatment with a retinoid antagonist reduced it. Interestingly, overexpression of retinoic acid receptor gamma (RARgamma) strongly inhibited Wnt/beta-catenin signaling in retinoid-free cultures, whereas small interfering RNA-mediated silencing of endogenous RARgamma expression strongly increased it. Small interfering RNA-mediated silencing of RARalpha or RARbeta had minimal effects. Co-immunoprecipitation and two-hybrid assays indicated that RARgamma interacts with beta-catenin and induces dissociation of beta-catenin from lymphoid enhancer factor in retinoid-free cultures. The N-terminal domain (AF-1) of RARgamma but not the C-terminal domain (AF-2) was required for association with beta-catenin, whereas both AF-1 and AF-2 were necessary for inhibition of beta-catenin transcriptional activity. Taken together, our data indicate that the Wnt and retinoid signaling pathways do interact in chondrocytes, and their cross-talks and cross-regulation play important roles in the regulation of cartilage matrix homeostasis.

Interhemispheric Modulation Induced by Cortical Stimulation and Motor Training

Interhemispheric inhibition might be a beneficial cortico-cortical interaction, but also might be maladaptive in people with neurological disorders. One recently revisited technique that has been shown to be effective in improving motor function in people with stroke using interhemispheric modulation is transcranial direct current stimulation (tDCS).

Sleep Triggered by an Immune Response in Drosophila is Regulated by the Circadian Clock and Requires the NFkappaB Relish

Immune challenge impacts behavior in many species. In mammals, this adaptive behavior is often manifested as an increase in sleep. Sleep has therefore been proposed to benefit the host by enhancing immune function and thereby overcome the challenge. To facilitate genetic studies on the relationship between sleep and immune function, we characterized the effect of the immune response on sleep in Drosophila melanogaster. Behavioral features of sleep as well as the innate immune response signaling pathways are well characterized in flies and are highly conserved in mammals.

Endogenous Retinoids in Mammalian Growth Plate Cartilage: Analysis and Roles in Matrix Homeostasis and Turnover

The growth plate contains resting and proliferating chondrocytes in its upper zones (UGP) and maturing and hypertrophic chondrocytes in its lower zones (LGP), but the mechanisms by which it operates to sustain skeletal growth are not fully clear. Retinoid signaling was previously found to be nearly absent in UGP, but to be much stronger in LGP coincident with hypertrophy, extracellular matrix turnover and endochondral bone formation. To determine whether such distinct signaling levels and phenotypic events reflect different endogenous retinoid levels, the upper two-thirds and lower one-third of rabbit rib growth plates were microsurgically isolated and processed for ultrasensitive retinoid LC-tandem MS quantification. Indeed, the UGP samples contained only about a 0.6 nm concentration of all-trans-retinoic acid (atRA) that is the most active natural retinoid in tissues, whereas LGP samples contained nearly 3-fold higher atRA levels (about 1.8 nM). Perichondrium was quite rich in atRA (about 4.9 nM). Interestingly, the levels of retinol, the major but inactive atRA precursor, were similar in all tissues (1.1-1.6 μM), suggesting that the distinct atRA levels in UGP and LGP reflect different retinoid anabolic capacity. Indeed, RALDH2 and CRABP1 transcript levels were much higher in LGP than UGP samples. To determine the minimum effective atRA concentration, chondrogenic cells transfected with a retinoic acid response element (RARE)-luc reporter plasmid were treated with different concentrations of exogenous atRA (0-100 nM). About 3 nm atRA was needed to elicit appreciable RARE-luc reporter activity and to decrease proteoglycan synthesis and activity of an aggrecan enhancer reporter plasmid. In sum, the data indicate that (i) the endogenous levels of atRA are significantly higher in hypertrophic than upper zones of growth plate; (ii) such difference likely reflects distinct retinoid anabolic capacity; and (iii) importantly, atRA levels in hypertrophic portion are within effective ranges to elicit retinoid signaling and action, but those in upper zones are not.

Patient Management After Noninvasive Cardiac Imaging Results From SPARC (Study of Myocardial Perfusion and Coronary Anatomy Imaging Roles in Coronary Artery Disease)

This study examined short-term cardiac catheterization rates and medication changes after cardiac imaging.

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