EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.

Improvement of a test-chamber for behavioral studies on adult females of Aedes aegypti (Linnaeus) (Diptera, Culicidae)

A test-chamber (K&L-Chamber) made of cardboard and acrylic plastic, and consisting in four sections (A, B, C and D) was developed by Klowden & Lea (1978) for Aedes aegypti host-seeking behavior studies. Later, Foster & Lutes (1985) also used an identical chamber to successfully evaluate the efficacy of electronic repellers. It was described here a modified K&L-Chamber for behavioral studies of Ae. aegypti adults. The chamber was made in polystyrene, consisting of three sections (A, B and C) and using a human hand and a fluorescent lamp as stimulus to attract the mosquitoes. The suitability of the present test-chamber was validated assaying 80 replicates and releasing 10 Ae. aegypti females in each replicate. The females were released in the section A and allowed to fly to the section C. A mean of 96.0% (s.e. 0.213) Ae. aegypti females successfully reached section C. The present test-chamber is cheaper and easier to handle and as efficient as K&L-Chamber, when compared to Foster & Lutes (1978) that noticed 93.8% of Ae. aegypti reaching the trap section.

D-3 2005-1 Dependent Adult Abuse Report, January-June 2005

Dependent Adult Abuse Report January 2005 – June 2005 Reported Dependent Adult Abuse by County

D-3 2006-1 Dependent Adult Abuse Report, January - June 2006

D-3 Dependent Adult Abuse Report

D-3 2007-1 Dependent Adult Abuse Report, January - June 2007

D-3 Dependent Adult Abuse Report

D-3 2007-2 Dependent Adult Abuse Report, July - December 2007

D-3 Dependent Adult Abuse Report

A new alternative method for testing skin irritation using a human skin model: a pilot study.

Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (Episkin®, Epiderm®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (Epiderm®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably evaluate reproducibility.

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions.

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.

D-3 2008-1 Dependent Adult Abuse Report, January - June 2008

D-3 Dependent Adult Abuse Report

Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform.

BACKGROUND: Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. METHODS AND RESULTS: In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Long-term expansion of transplantable human fetal liver hematopoietic stem cells

Hematopoietic stem cells (HSCs), with their dual ability for self-renewal and multilineage differentiation, constitute an essential component of hematopoietic transplantations. Human fetal liver (FL) represents a promising alternative HSC source, and we previously reported simple culture conditions allowing long-term expansion of FL hematopoietic progenitors. In the present study, we used the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenotransplantation assay to confirm that human FL is rich in NOD/SCID-repopulating cells (SRCs) and to show that these culture conditions repeatedly maintained short- and long-term SRCs from various FL samples for at least 28 days. Quantitative limited dilution analysis in NOD/SCID mice demonstrated for the first time that a 10- to over a 100-fold net expansion of FL SRCs could be achieved after 28 days of culture. The efficiency of this culture system may lead to an increase in the use of FL as a source of HSCs for transplantation in adult patients, as previously demonstrated with umbilical cord blood under different culture conditions.

Assessment of speed of human locomotion using a differential satellite global positioning system.

PURPOSE: The objective was to explore whether a satellite-based navigation system, global positioning system used in differential mode (DGPS), could accurately assess the speed of running in humans. METHODS: A subject was equipped with a portable GPS receptor coupled to a receiver for differential corrections, while running outdoors on a straight asphalt road at 27 different speeds. Actual speed (reference method) was assessed by chronometry. RESULTS: The accuracy of speed prediction had a standard deviation (SD) of 0.08 km x h(-1) for walking, 0.11 km x h(-1) for running, yielding a coefficient of variation (SD/mean) of 1.38% and 0.82%, respectively. There was a highly significant linear relationship between actual and DGPS speed assessment (r2 = 0.999) with little bias in the prediction equation, because the slope of the regression line was close to unity (0.997). CONCLUSION: the DGPS technique appears to be a valid and inconspicuous tool for "on line" monitoring of the speed of displacement of individuals located on any field on earth, for prolonged periods of time and unlimited distance, but only in specific environmental conditions ("open sky"). Furthermore, the accuracy of speed assessment using the differential GPS mode was improved by a factor of 10 as compared to non-differential GPS.

GPS analysis of human locomotion: further evidence for long-range correlations in stride-to-stride fluctuations of gait parameters.

During free walking, gait is automatically adjusted to provide optimal mechanical output and minimal energy expenditure; gait parameters, such as cadence, fluctuate from one stride to the next around average values. It was described that this fluctuation exhibited long-range correlations and fractal-like patterns. In addition, it was suggested that these long-range correlations disappeared if the participant followed the beep of metronome to regulate his or her pace. Until now, these fractal fluctuations were only observed for stride interval, because no technique existed to adequately analyze an extended time of free walking. The aim of the present study was to measure walking speed (WS), step frequency (SF) and step length (SL) with high accuracy (<1 cm) satellite positioning method (global positioning system or GPS) in order to detect long-range correlations in the stride-to-stride fluctuations. Eight participants walked 30 min under free and constrained (metronome) conditions. Under free walking conditions, DFA (detrended fluctuation analysis) and surrogate data tests showed that the fluctuation of WS, SL and SF exhibited a fractal pattern (i.e., scaling exponent alpha: 0.5 < alpha < 1) in a large majority of participants (7/8). Under constrained conditions (metronome), SF fluctuations became significantly anti-correlated (alpha < 0.5) in all participants. However, the scaling exponent of SL and WS was not modified. We conclude that, when the walking pace is controlled by an auditory signal, the feedback loop between the planned movement (at supraspinal level) and the sensory inputs induces a continual shifting of SF around the mean (persistent anti-correlation), but with no effect on the fluctuation dynamics of the other parameters (SL, WS).

Neurophysiological origin of human brain asymmetry for speech and language.

The physiological basis of human cerebral asymmetry for language remains mysterious. We have used simultaneous physiological and anatomical measurements to investigate the issue. Concentrating on neural oscillatory activity in speech-specific frequency bands and exploring interactions between gestural (motor) and auditory-evoked activity, we find, in the absence of language-related processing, that left auditory, somatosensory, articulatory motor, and inferior parietal cortices show specific, lateralized, speech-related physiological properties. With the addition of ecologically valid audiovisual stimulation, activity in auditory cortex synchronizes with left-dominant input from the motor cortex at frequencies corresponding to syllabic, but not phonemic, speech rhythms. Our results support theories of language lateralization that posit a major role for intrinsic, hardwired perceptuomotor processing in syllabic parsing and are compatible both with the evolutionary view that speech arose from a combination of syllable-sized vocalizations and meaningful hand gestures and with developmental observations suggesting phonemic analysis is a developmentally acquired process.