Electrophoresis of positioned nucleosomes

We present in this paper an original approach to compute the electrophoretic mobility of rigid nucleo-protein complexes like nucleosomes. This model allows to address theoretically the influence of complex position along DNA, as well as wrapped length of DNA on the electrophoretic mobility of the complex. The predictions of the model are in qualitative agreement with experimental results on mononucleosomes assembled on short DNA fragments (<400bp). Influence of additional experimental parameters like gel concentration, ionic strength, effective charges is also discussed in the framework of the model, and is found to be qualitatively consistent with experiments when available. Based on the present model, we propose a simple semi-empirical formula describing positioning of nucleosomes as seen through electrophoresis.Comment: to appear in Biophys. J. 29 page

CD36 plays an important role in the clearance of oxLDL and associated age-dependent sub-retinal deposits

Age-related macular degeneration (AMD) represents the major cause of vision loss in industrialized nations. Laminar deposits in Bruch's membrane (BM) are among the first prominent histopathologic features, along with drusen formation, and have been found to contain oxidized lipids. Increases in concentrations of oxidized LDL (oxLDL) in plasma are observed with age and high fat high (HFHC) cholesterol diet. CD36 is the principal receptor implicated in uptake of oxLDL, and is expressed in the retinal pigment epithelium (RPE). We determined if CD36 participates in oxLDL uptake in RPE and correspondingly in clearance of sub-retinal deposits. Uptake of oxLDL by RPE in vitro and in vivo was CD36-dependent. CD36 deficiency in mice resulted in age-associated accumulation of oxLDL and sub-retinal BM thickening, despite fed a regular diet. Conversely, treatment of HFHC-fed ApoE null mice with a CD36 agonist, EP80317 (300 μg/kg/day), markedly diminished thickening of BM, and partially preserved (in part) photoreceptor function. In conclusion, our data uncover a new role for CD36 in the clearance of oxidized lipids from BM and in the prevention of age-dependent sub-retinal laminar deposits

Risks factors for significant injury after geriatric falls

Elderly falls are a healthcare epidemic. We aimed to identify risk factors of serious falls by linking data on functional status from the Global Longitudinal Study of Osteoporosis in Women (GLOW) and our institutional trauma registry. 124 of 5,091 local women enrolled in GLOW were evaluated by our trauma team for injuries related to a fall during the study period. Median injury severity score was 9. The most common injuries were intertrochanteric femur fracture (n = 25, 9.8%) and skin contusion/hematoma to face (n = 12, 4.7%). Injured women were older than the uninjured cohort (median 80 versus 68 years), more likely to have cardiovascular disease and osteoarthritis, and less likely to have high cholesterol. Prospectively collected Short Form 36 (SF-36) baseline activity status revealed greater limitation in all assessed activities in women evaluated for fall-related injuries in our trauma center. In multivariable analysis, age (per 10 year increase) and two or more self-reported falls in the baseline survey were the strongest predictors of falling (both HR 2.4, p &lt;0.0001 and p&lt;0.001 respectively), followed by history of osteoarthritis (HR 1.6, p= 0.01). Functional status was no longer associated with risk of fall when adjusting for these factors.Functional status appears to be a surrogate marker for frailty. With the aging of the US population and long lifespan of American women, this finding has important implications for both fall prevention strategies and research intended to better understand why aging women fall as burdensome validated metrics may not be the best indicators of fall risk. The authors have no conflict of interests to declare. Funding statement: This work was partially supported by grants from NIH (8KL2TR000160-03), AHRQ (R01HS22694), and PCORI (ME-1310-07682) to HS

Oxygen induced retinopathy in the neonatal rat: the effects of age, strain and therapeutic intervention on retinal structure and function

Retinopathy of prematurity (ROP) is a potentially blinding retinal disorder that affects small prematurely born infants which results from the combination of the immature retina and the high level of oxygen (hyperoxia) needed to keep these infants alive. A rat model of oxygen induced retinopathy (OIR) was developed to better understand what occurs in premature infants that are exposed to postnatal hyperoxia and previous studies have demonstrated that this model bears a striking resemblance to the human form of ROP, at least in its vascular component. The purpose of this study was to investigate beyond the retinal vasculature in order to better understand the functional [electroretinogram (ERG)] and cytoarchitectural (histology and immunohistochemistry) manifestations of this disease.While structural and functional consequences of OIR in the albino rat had previously been evaluated in mature rats (at postnatal days 30 and 60, for example), the sequence of events leading up to these changes could only be inferred. Consequently, the purpose of our first study was to further characterize the early manifestations of this disease process, namely: 1- to evaluate changes in retinal vasculature throughout exposure to hyperoxia and, 2- to examine how early after the cessation of oxygen exposure we can demonstrate changes in retinal structure and function. Findings suggest that while the vascular growth process could repair itself while still subjected to a hyperoxic environment (from P0-12 and P0-14, for example), irreversible changes in retinal ultrastructure and function could still be evidenced, and are documented immediately following the cessation of the hyperoxic regimen (Dorfman et al., 2008).It has been suggested that free radicals may be the source of the pathologic effects described above. Given that trolox C, a water-soluble analogue of vitamin E, was previously shown to limit the vasculopathy component of OIR, the aim of our next study was to determine whether it could prevent the functional and structural consequences of OIR. While trolox C could limit the damage intrinsic to the rat model of OIR, it could not entirely prevent it from occurring. This suggests that either OIR is not solely caused by the action of free radicals or that trolox C is inadequate in treating all aspects of OIR (Dorfman et al., 2006).We next sought to compare our findings using trolox C with those obtained using the pigmented Long Evans (LE) rat in which melanin is thought to play an antioxidant role. Our results show that melanin did not exert the predicted protective effect on retinal structure and function, where LE rats were affected earlier on and more drastically than albino Sprague Dawley rats following hyperoxic exposure (Dorfman et al., 2009). Specific markers for rod and cone bipolar cells (PKC-α and recoverin, respectively), amacrine cells (parvalbumin) and horizontal cells (calbindin) reveal that OIR is primarily an inner retinal disorder, the earliest consequences of which include cell death in the inner retina (TUNEL staining) and synaptic retraction in the OPL (synaptophysin) (Dorfman et al., in preparation).Collectively, our results suggest that the rat model of OIR is not only useful for studying retinal vasculature, but can also be used to further elucidate the functional and structural sequelae that likely resemble the human form of ROP. Our ability to demonstrate persistant functional and cytoarchitectural damage despite vascular repair would suggest the importance of examining beyond the fundus in ROP patients, should these features also apply to humans. ROP is one of several types of proliferative retinopathies characterized by retinal ischemia followed by abnormal vessel growth. Studies such as these will undoubtedly be instrumental in a better understanding of these types of retinopathies and in the derivation of new therapeutic avenues aimed at controlling them.La rétinopathie du prématuré (RDP) est une maladie rétinienne qui potentiellement aboutie à la cécité des enfants de faible poids nés prématurément et qui est causée par la combinaison de l'immaturité de la rétine et du niveau élevé d'oxygène (hyperoxie) essentiel pour garder ces nouveau-nés en vie. Afin de mieux saisir l'effet de l'hyperoxie postnatale chez ces bébés prématurés, un modèle animal de rétinopathie induit par l'oxygène (RIO) a été développé chez le rat et a démontré une forte ressemblance à la forme humaine de la RDP, du moins dans sa composante vasculaire. Notre but était d'examiner au-delà de la vascularisation rétinienne afin de mieux comprendre les manifestations fonctionnelles [électrorétinogramme (ERG)] et cytoarchitecturelles (histologie et immunohistochimie) de cette maladie.L'évaluation antérieure de la fonction et de la structure rétinienne chez les rats albinos matures (par exemple à 30 et 60 jours postnatals) de la RIO a uniquement permis de déduire la chaîne d'évènements menant à ces changements. Par conséquent, la première étude consistait à caractériser les signes hâtifs de la RIO, pour: 1 - évaluer la vascularisation rétinienne durant l'exposition et, 2 - savoir quand, au plus tôt, après l'exposition les changements de structure et de fonction peuvent être observés. Les résultats suggèrent que la croissance vasculaire pourrait se restaurer durant le stress hyperoxique (expositions de P0-12 et de P0-14, par exemple), malgré la présence des changements irréversibles de l'ultrastructure rétinienne et sa fonction survenant immédiatement après la fin du régime hyperoxique (Dorfman et al., 2008).Les radicaux libres ont été suggérés d'être à l'origine des effets pathologiques décrits ci-dessus. Étant donné que trolox C, un analogue hydrosoluble de la vitamine E, a été démontré de limiter la composante vasculaire de la RIO, le but de l'étude suivante était de voir s'il pouvait aussi prévenir les séquelles fonctionnelles et structurelles. Nos résultats montrent que trolox C représente une alternative thérapeutique valide limitant les dommages, mais il n'empêche pas la totalité des dégâts rétiniens causés par l'oxygène. Ceci suggère que la RIO n'est pas juste provoquée par l'action des radicaux libres ou que le trolox C est inadéquat pour traiter tous les aspects de la RIO (Dorfman et al., 2006).Nous avons ensuite comparé nos résultats de trolox C à ceux obtenus avec le rat pigmenté Long Evans (LE) dans lequel la mélanine est présumée de jouer un rôle antioxydant. Nos données montrent que la mélanine n'exerce pas l'effet protecteur prévu sur la structure et la fonction de la RIO, où les rats LE ont été affectés plus tôt et plus gravement que les rats albinos Sprague Dawley suivant l'exposition à l'oxygène (Dorfman et al., 2009). Le marquage spécifique pour les cellules bipolaires de bâtonnet et de cône (PKC-α et recoverin, respectivement), les cellules amacrine (parvalbumine) et les cellules horizontales (calbindine) indiquent que la RIO est une maladie de la rétine interne définie par une mort cellulaire à ce niveau (TUNEL) et par la rétraction synaptique dans la couche plexiforme externe (synaptophsine) (Dorfman et al., manuscrit en préparation).En conclusion, nos résultats suggèrent que le modèle de rat de la RIO est non seulement utile pour étudier la vascularisation rétinienne, mais peut aussi être employé pour élucider les conséquences fonctionnelles et structurelles pouvant ressembler au modèle humain de la RDP. Notre capacité de démontrer les dégâts fonctionnels et cytoarchitecturels persistants en dépit de la réparation vasculaire suggérerait l'importance de l'examen approfondi chez les patients avec la RDP. La RDP est une forme des nombreuses rétinopathies prolifératives. Ces genres d'études seront certainement utiles pour une meilleure caractérisation de ces types de rétinopathies et dans la création de nouvelles méthodes thérapeutiques visant à mieux les contrôler

Monitoring Nantucket Harbor

Monitoring and maintaining the quality of Nantucket Harbor is the responsibility of the Nantucket Department of Natural Resources as they attempt to preserve the harbor’s aquatic life and economy. The goal of this project was to compile all available data on water quality regarding Nantucket Harbor, make observations in trends regarding the harbor’s health, and make recommendations to better record and communicate information on water quality. This goal was met by specifically analyzing nitrates and nitrogen inputs into the harbor and addressing the need for more detailed monitoring of water quality factors

Immunohistochemical evidence of synaptic retraction, cytoarchitectural remodeling, and cell death in the inner retina of the rat model of Oygen-Induced Retinopathy (OIR)

Purpose. Postnatal exposure to hyperoxia destroys the plexiform layers of the neonatal rat retina, resulting in significant electroretinographic anomalies. The purpose of this study was to identify the mechanisms at the origin of this loss. Methods. Sprague-Dawley (SD) and Long Evans (LE) rats were exposed to hyperoxia from birth to postnatal day (P) 6 or P14 and from P6 to P14, after which rats were euthanatized at P6, P14, or P60. Results. At P60, synaptophysin staining confirmed the lack of functional synaptic terminals in SD (outer plexiform layer [OPL]) and LE (OPL and inner plexiform layer [IPL]) rats. Uneven staining of ON-bipolar cell terminals with mGluR6 suggests that their loss could play a role in OPL thinning. Protein kinase C(PKC)-α and recoverin (rod and cone ON-bipolar cells, respectively) showed a lack of dendritic terminals in the OPL with disorganized axonal projections in the IPL. Although photoreceptor nuclei appeared intact, a decrease in bassoon staining (synaptic ribbon terminals) suggests limited communication to the inner retina. Findings were significantly more pronounced in LE rats. An increase in TUNEL-positive cells was observed in LE (inner nuclear layer [INL] and outer nuclear layer [ONL]) and SD (INL) rats after P0 to P14 exposure (425.3%, 102.2%, and 146.3% greater than control, respectively [P < 0.05]). Conclusions. Results suggest that cell death and synaptic retraction are at the root of OPL thinning. Increased TUNEL-positive cells in the INL confirm that cells die, at least in part, because of apoptosis. These findings propose a previously undescribed mechanism of cell death and synaptic retraction that are likely at the origin of the functional consequences of hyperoxia.Supported by Canadian Institutes of Health Research Grant MOP-13383; FRSQ-Réseau Vision; McGill University-Montreal Children's Hospital Research Institute; Ministerio de Ciencia e Innovación Grant BFU2009-07793/BFI; RETICS Grant RD07/0062/0012; Fundaluce; ONCE; Fundación Médica Mutua Madrileña (NC); and FIS Grant PS09/01854 (IP)

Hyperoxic exposure leads to nitrative stress and ensuing microvascular degeneration and diminished brain mass and function in the immature subject.

BACKGROUND AND PURPOSE: Neonates that survive very preterm birth have a high prevalence of cognitive impairment in later life. A common factor detected in premature infants is their postnatal exposure to high oxygen tension relative to that in utero. Hyperoxia is known to elicit injury to premature lung and retina. Because data on the exposure of the brain to hyperoxia are limited, we studied the effects of high oxygen on this tissue. METHODS: Rat pups were exposed from birth until day 6 to 21% or 80% O(2). Cerebral vascular density was quantified by lectin immunohistochemistry. Immunoblots for several proteins were performed on brain extracts. We assessed cerebral functional deficits by visual evoked potentials. RESULTS: Exposure of pups to hyperoxia leads to cerebral microvascular degeneration, diminished brain mass, and cerebral functional deficits. These effects are preceded by an upregulation of endothelial nitric oxide synthase (eNOS) in cerebral capillaries and a downregulation of Cu/Zn superoxide dismutase (SOD). The imbalance in nitric oxide (NO) production and antioxidant defenses favors the formation of nitrating agents in the microvessels revealed by increased nitrotyrosine (3-nt) immunoreactivity and decreased expression of NF-kappaB and the dependent vascular endothelial growth factor receptor 2. NOS inhibitors and eNOS deletion as well as an SOD mimetic (CuDIPS) restore vascular endothelial growth factor receptor-2 levels and nearly abolish the vasoobliteration. NOS inhibitors and SOD mimetic also prevent O(2)-induced diminished brain mass and functional deficit. CONCLUSIONS: Data identify NO and nitrating agents as major mediators of cerebral microvascular damage, ensuing impaired brain development and function in immature subjects exposed to hyperoxia