Prolactin receptor signaling is essential for perinatal brown adipocyte function: a role for insulin-like growth factor-2.

BACKGROUND: The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARgamma2, its coactivator PGC-1alpha, uncoupling protein 1 (UCP1) and the beta3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2. CONCLUSIONS: Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia.

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.

BACKGROUND: Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). METHODS AND FINDINGS: Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. CONCLUSIONS: In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

Quantifiable biomarkers of normal aging in the Japanese medaka fish (Oryzias latipes).

BACKGROUND: Small laboratory fish share many anatomical and histological characteristics with other vertebrates, yet can be maintained in large numbers at low cost for lifetime studies. Here we characterize biomarkers associated with normal aging in the Japanese medaka (Oryzias latipes), a species that has been widely used in toxicology studies and has potential utility as a model organism for experimental aging research. PRINCIPAL FINDINGS: The median lifespan of medaka was approximately 22 months under laboratory conditions. We performed quantitative histological analysis of tissues from age-grouped individuals representing young adults (6 months old), mature adults (16 months old), and adults that had survived beyond the median lifespan (24 months). Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits and in the number of TUNEL-positive apoptotic cells. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in senescence-associated β-galactosidase. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain. SIGNIFICANCE: The results provide a set of markers that can be used to trace the process of normal tissue aging in medaka and to evaluate the effect of environmental stressors.

Drinking from arboreal water sources by mantled howling monkeys (Alouatta palliata Gray).

Despite occasional trips to the ground and feeding in trees whose canopies touched the river, mantled howling monkeys were never seen to drink from any ground water. Drinking from arboreal cisterns was observed, but only during the wet season (meteorologically the less stressful season but phenologically the more stressful season). The lack of sufficient new leaves during the wet season forced the howlers to ingest more mature leaves which contained significantly less water. To compensate for the lowered amount of water in their food, the monkeys utilized arboreal water cisterns. The cisterns dried up during the dry season, but the howlers maintained their water balance by altering their time of actiivity and selecting a diet comprised largely of succulent new leaves. The effect of plant-produced secondary compounds on drinking also was discussed.

Consumption of cyanogenic bamboo by a newly discovered species of bamboo lemur

Three species of bamboo‐eating lemurs were found to be sympatric in the southeastern rain forests of Madagascar. Sympatric species generally differ in habitat utilization or diet, but these three closely related bamboo lemurs lived in the same habitat and all ate bamboo. Behavioral observation revealed that they did select different parts of the bamboo, and chemical analyses confirmed that there was a difference in the secondary compound content present in those selections. The growing tips of Cephalostachyum ef uiguieri selected by the golden bamboo lemur (Hapalemuraureus) contained 15 mg of cyanide per 100 g fresh weight bamboo while the leaves of C. perrieri selected by the gentle bamboo lemur (H. griseus)and the mature culms of C. cf uiguieri selected by the greater bamboolemur (H. simus) did not contain cyanide. Since each individual golden bamboo lemur ate about 500 g of bamboo per day, they daily ingestedabout 12 times the lethal dose of cyanide. The mechanism by which this small primate avoids the acute and chronic symptoms of cyanide poisioning is unknown. Copyright © 1989 Wiley‐Liss, Inc., A Wiley Company

TRANSCENDING FACH: A SEARCH FOR IDENTITY INSIDE AND OUT OF MEZZO-SOPRANO REPERTOIRE

The German Fach system is a tool to classify voices in classical singing. This dissertation comprises three different programs that reflect my search for identity as a mezzo-soprano and my desire to transcend the limitations of Fach. The three programs, all presented at The Clarice Performing Arts Center, contain repertoire written for male and female voices thus allowing me to explore areas outside of the mezzo-soprano Fach, gain a better understanding of the Fach system and guide me as I strive to become a more mature performer. In my first program, I sang the role of Sesto, a role that was composed originally for a castrate, in the opera La Clemenza di Tito by W.A. Mozart. The Maryland Opera Studio production took place April 30, May 2,4&6,2003. Performing this gender-bending role provided an experience of physical behavior from the male view point along with the demands of coloratura singing. Program two (November 30,2004) contained the song cycle Dichterliebe by Robert Schumann and songs by Ludwig van Beethoven, Franz Schubert and Felix Mendelssohn, which are usually sung by male voices. This program experimented with extended range, tessitura and a gender-bending performance in the art song arena. 8 In program three (April 21 &23,2005), I sang the contralto role of Cornelia from Giulio Cesare in Egitto by George Frederic Handel. The role of Cornelia is psychologically complex, expressing emotions such as love, melancholy, rage, malice, joy and fear. To convey these emotions a voice needs warmth and darkness of quality. Although the range is close to that of the mezzo-soprano, Handel wrote Cornelia for contralto voice because he wanted a dark timbre and this role allowed me to develop my lower register and manage suitable ornamentations. The programs are documented in a digital format available on compact disc and are accompanied by the oral presentation at the defense of this dissertation.

Four-decade responses of soil trace elements to an aggrading old-field forest: B, Mn, Zn, Cu, and Fe.

In the ancient and acidic Ultisol soils of the Southern Piedmont, USA, we studied changes in trace element biogeochemistry over four decades, a period during which formerly cultivated cotton fields were planted with pine seedlings that grew into mature forest stands. In 16 permanent plots, we estimated 40-year accumulations of trace elements in forest biomass and O horizons (between 1957 and 1997), and changes in bioavailable soil fractions indexed by extractions of 0.05 mol/L HCl and 0.2 mol/L acid ammonium oxalate (AAO). Element accumulations in 40-year tree biomass plus O horizons totaled 0.9, 2.9, 4.8, 49.6, and 501.3 kg/ha for Cu, B, Zn, Mn, and Fe, respectively. In response to this forest development, samples of the upper 0.6-m of mineral soil archived in 1962 and 1997 followed one of three patterns. (1) Extractable B and Mn were significantly depleted, by -4.1 and -57.7 kg/ha with AAO, depletions comparable to accumulations in biomass plus O horizons, 2.9 and 49.6 kg/ha, respectively. Tree uptake of B and Mn from mineral soil greatly outpaced resupplies from atmospheric deposition, mineral weathering, and deep-root uptake. (2) Extractable Zn and Cu changed little during forest growth, indicating that nutrient resupplies kept pace with accumulations by the aggrading forest. (3) Oxalate-extractable Fe increased substantially during forest growth, by 275.8 kg/ha, about 10-fold more than accumulations in tree biomass (28.7 kg/ha). The large increases in AAO-extractable Fe in surficial 0.35-m mineral soils were accompanied by substantial accretions of Fe in the forest's O horizon, by 473 kg/ha, amounts that dwarfed inputs via litterfall and canopy throughfall, indicating that forest Fe cycling is qualitatively different from that of other macro- and micronutrients. Bioturbation of surficial forest soil layers cannot account for these fractions and transformations of Fe, and we hypothesize that the secondary forest's large inputs of organic additions over four decades has fundamentally altered soil Fe oxides, potentially altering the bioavailability and retention of macro- and micronutrients, contaminants, and organic matter itself. The wide range of responses among the ecosystem's trace elements illustrates the great dynamics of the soil system over time scales of decades.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish.

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.

THE PIANO MUSIC OF ROBERT SCHUMANN (1810-1856) AND JOHANNES BRAHMS (1833- 1897): A SURVEY OF CONCERTI, VARIATIONS, AND CHARACTER PIECES

Robert Schumann (1810-1856) and Johannes Brahms (1833-1897), in some ways Robert Schumann's artistic descendant, are the most important and representative German piano composers during the Romantic period. Schumann was already a mature and established musician in 1853 when he first met the young Brahms and recognized his talents, an encounter that had a long-lasting affect on the lives and careers of both men. After Schumann’s mental breakdown and death, Brahms maintained his admiration of Schumann’s music and preserved an intimate relationship with Clara Schumann. In spite of the personal and musical closeness of the two men, Schumann’s music is stylistically distinct from that of Brahms. Brahms followed traditions from Baroque and Classical music, and avoided using images and expressive titles in his music. Brahms extraordinarily intermingled earlier musical forms with multicolored tones of German Romanticism. In contrast, Schumann saw himself as a radical composer devoted to personal emotionalism and spontaneity. He favored programmatic titles for his character pieces and extra-musical references in his music. While developing their own musical styles as German Romantic composers, Schumann and Brahms both utilized the piano as a resourceful tool for self-realization and compositional development. To investigate and compare the main characteristics of Schumann and Brahms’s piano music, I looked at three genres. First, in the category of the piano concerto, I chose two major Romantic works, Schumann’s A minor concerto and Brahms’s B-flat major concerto. Second, for the category of piano variations I included two sets by Brahms because the variation framework was such an important vehicle for him to express his musical thoughts. Schumann’s unique motivic approach to variation is displayed vividly in his character-piece cycle Carnaval. Third, the category of the character piece, perhaps the favorite medium of Romantic expression at the piano, is shown by Schumann’s Papillons and Brahms’s sets of pieces Op.118 and Op.119. This performance dissertation consists of three recitals performed in the Gildenhorn Recital Hall at the University of Maryland, College Park. These recitals are documented on compact disc recordings that are housed within the University of Maryland Library System.

The Evolution of Rachmaninov's Pianistic Style from 1892 to 1941 as Seen in Selected Original Works and Transcriptions for Piano

Unlike some other major composer-pianists (Franz Liszt and Alexander Scriabin being the most notable examples), Sergei Rachmaninov did not experience any dramatic changes in his compositional style; one can, however, notice certain stylistic differences between piano works composed during different periods of his long creative life. This dissertation argues that a clear evolution of Rachmaninov's pianistic style through his three creative periods can be traced by examining a selection of his compositions, from his first significant cycle for piano, 5 Marceaux de fantaisie, Op. 3 (1892), all the way through to his last piano work, a transcription of P.I.Tchakovsky's Lullaby, Op. 16, No. 1 (1941). Rachmaninov's life as a composer can be divided into three periods. His early period was ended abruptly by the disastrous premiere ofhis First Symphony in 1897, which caused a deep psychological crisis in the life of the young composer. Piano works of this period are often characterized by relatively simple homophonic texture, when Rachmaninov was clearly influenced by some of his Russian predecessors, most notably Tchaikovsky. His second and most productive period, also known as the period of"Russian maturity," started in 1900, when he began working on the Second Suite for two pianos, Op. 17, and the Second Concerto, Op. 18; this phase ended with the Russian Bolshevik revolution of 1917. Works of this time exhibit a mature style of piano writing, with rich, virtuosic - often considered excessive by many critics - texture and ever-increasing use of chromatic harmonies. Rachmaninov's works of the third period are limited in number owing to the composer's preoccupation with a career as pianist. Original works for piano now give way to revisions of earlier compositions and transcriptions: Rachmaninov's piano writing becomes more efficient and economical without losing any of its virtuosic brilliance. This dissertation project examines in detail, over the course of three piano recitals, a variety of works composed during the "Russian maturity" period, from several Preludes from Op. 23 (1903), the first major cycle for solo piano of the period, to 9 Etudes-Tableaux, Op.39 (1917), the last one; the early period is represented by 3 of 5 Marceaux defantaisie, Op. 3 (1892), while the late period is shown through several piano transcriptions and revised versions of the remaining 2 pieces from Op. 3.

SCHUBERT’S WINTERREISE AND THE PIANO TRIO IN Eb; TWO REMARKABLE WORKS FROM ONE REMARKABLE YEAR – 1827: A RECORDING PROJECT

During Franz Schubert’s penultimate year of 1827, he produced two profoundly important and mature works that are the focus of this recording project. The works are, in chronological order: • Winterreise (cycle of 24 songs on the poetry of Wilhelm Müller, 1794-1827) • Piano Trio in Eb Major, Op. 100, D. 929 A unique feature of the project is to present Winterreise in two poetic orders: as traditionally performed and published by Schubert, and in the final ordering published by the poet. The program notes accompanying the dissertation’s three compact discs have extensive information as well as comparative tables of Müller’s and Schubert’s final ordering of the cycle. There are significant differences in ordering, and ultimately the listener will determine which is more dramatically satisfying. Dark melancholy is the central emotion in Winterreise, which Schubert composed at various times throughout 1827 in a mood of corresponding gloom and distress. By contrast, the summer and fall of that year produced, in quick succession, the two glowing and remarkable Piano Trios in Bb and Eb, the second of which is included on these compact discs. The contrast between the trios and Winterreise follows the outward circumstances of Schubert’s life and health, a pattern of sorrow and later consolation and elation. The sound recordings for this dissertation recording project are available on three compact discs that can be found in the Digital Repository at the University of Maryland (DRUM). Winterreise was recorded in August 2009, at the University of Baltimore recital hall in Baltimore, Maryland with University of Maryland Professor François Loup. The trio, recorded in live performance in Baltimore in the spring of 2010, features two members of the Baltimore Symphony Orchestra: Qing Li, B.S.O. principal second violin, and Bo Li, B.S.O. section cellist.

Preliminary experience of ovarian tissue cryopreservation procedure: alternatives, perspectives and feasibility

Chemotherapy and radiotherapy induce premature ovarian failure in many patients treated for oncological or benign diseases. The present paper reviews the risk of developing premature ovarian failure according to the type of treatment and the different options to preserve fertility, focusing on the cryopreservation of ovarian tissue. This technique constitutes a promising approach to preserve the fertility of young patients and offers the advantage of storing a large number of follicles that could be subsequently transplanted or cultured in vitro to obtain mature oocytes. Based on 34 requests, from which 19 were performed, the feasibility of the ovarian cryopreservation procedure is evaluated. The medical and ethical approaches of this protocol are also discussed. Cryopreservation of ovarian tissue constitutes new hope for many patients, but must still be kept for selected cases, with a significant risk of premature ovarian failure after treatments such as bone marrow transplantation.

Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses.

Neonatal immaturity of the immune system is currently believed to generally limit the induction of immune responses to vaccine Ags and to skew them toward type 2 responses. We demonstrated here that Bordetella pertussis infection in very young infants (median, 2 mo old) as well as the first administration of whole-cell pertussis vaccine induces B. pertussis Ag-specific IFN-gamma secretion by the PBMC of these infants. IFN-gamma was secreted by both CD4(+) and CD8(+) T lymphocytes, and the levels of Ag-induced IFN-gamma secretion did not correlate with the age of the infants. Appearance of the specific Th-1 cell-mediated immunity was accompanied by a general shift of the cytokine secretion profile of these infants toward a stronger Th1 profile, as evidenced by the response to a polyclonal stimulation. We conclude that the immune system of 2-mo-old infants is developmentally mature enough to develop Th1 responses in vivo upon infection by B. pertussis or vaccination with whole-cell pertussis vaccines.